Eric Kool received his Ph.D. in Chemistry from Columbia University and did postdoctoral work in nucleic acids chemistry at Caltech. He started his career at the University of Rochester before moving to Stanford in 1999, where he is currently the George and Hilda Daubert Professor of Chemistry. He teaches Organic Chemistry and Chemical Biology to undergraduate and graduate students.

Academic Appointments

Honors & Awards

  • Breslow Award for Achievement in Biomimetic Chemistry, American Chemical Society (2015)
  • Dean’s Award for Distinguished Teaching, Stanford University (2015)
  • O. K. Rice Lectureship, University of North Carolina (2015)
  • Frontiers in Chemistry Distinguished Lecturer, Case Western Reserve University (2014)
  • Tarrant Distinguished Lectureship, University of Florida (2014)
  • O’Malley Lectureship, Boston College (2012)
  • Tortellotte Lectureship, Kalamazoo College (2010)
  • Hirschmann Lectureship, Oberlin College (2003)
  • Novartis Lecturer, Massachusetts Institute of Technology (2003)
  • Fellow of the AAAS, American Association for the Advancement of Science (2002)
  • Arthur C. Cope Scholar Award, American Chemical Society (2001)
  • Bernard Belleau Memorial Lecturer, McGill University (2001)
  • Dean’s Award for Distinguished Teaching, Stanford University (2001)
  • Pfizer Award, American Chemical Society (2000)
  • Alfred P. Sloan Foundation Fellow, Alfred P. Sloan Foundation (1994)
  • American Cyanamid Faculty Award, American Cyanamid (1994)
  • Army Young Investigator Award, Army Research Office (1993)
  • Camille and Henry Dreyfus Teacher - Scholar Award, Camille and Henry Dreyfus Foundation (1993)
  • Arnold & Mabel Beckman Foundation Young Investigator, Arnold & Mabel Beckman Foundation (1992)
  • Office of Naval Research Young Investigator Award, Office of Naval Research (1992)

Professional Education

  • PhD, Columbia University, Organic Chemistry, Biochemistry (1988)

Research & Scholarship

Current Research and Scholarly Interests

One major focus of our laboratory is the design, synthesis and study of unnatural DNA and RNA bases. These are used as tools for basic study of biochemical and biological mechanisms (see our work with "hydrophobic isosteres"), and as the basis for a new genetic system design (see "xDNA").

An area in which we make extensive use of designer DNA bases is in "DNA polyfluors", which are short segments of synthetic DNA in which the bases are replaced by fluorescent structures. These have been developed into a large palette of fluorescent labels for biology, offering benefits that current fluorescent labels do not have. They are also being built into sensors: short synthetically modified DNAs are being used to sense metal ions in water, and toxic gases in air. We are building arrays of fluorescent DNAs that can “smell” the metabolites that different bacteria emit, allowing us to distinguish disease-causing bacteria in a Petri dish. Our lab is also designing a broad array of multicolor sensors for different classes of enzymes, from esterases to proteases and DNA repair enzymes. These can function in living cells to report on biological activities there.

Our lab has been developing a new, functional genetic set that is orthogonal to the natural DNA system. Our design is based on expanded size (expanded DNA, or "xDNA"). We have shown that xDNA assembles into helices selectively, much like DNA, except that it is more stable and is also highly fluorescent. We have found polymerase enzymes that can copy bases of xDNA, and have shown that E. coli replication machinery can read the genetic information stored in xDNA. Very recently, we have begun to synthesize and study xRNA as well.

Our group also has an ongoing interest in finding simple and rapid ways to detect the cellular genetic mutations that cause cancer and drug resistance. Our approach is to use RNA-templated chemistry, in which chemically modified probes perform a fluorogenic reaction when they hybridize to their genetic target. Our laboratory was the first to use nucleic acid templated chemistry for detection of genetic sequences in solution, and the first to apply it in intact bacterial and human cells. New ways to improve this chemistry and monitor the output signal of these sensor molecules are under research. We are collaborating with clinical laboratories and physicians to test these molecules for applications in identifying pathogenic bacteria and in monitoring genetic changes in cells from patients with leukemias.

We have a general interest in the design of small-molecule probes and reagents that are useful in the study of cellular biomolecules. For example, we are developing cell-permeable reagents that can be used to map structure and contacts of RNAs in vivo. We are also developing tools for investigating RNA base modifications as well as DNA damage, and the enzymes that process these structural alterations. Finally, we are developing reagents for performing efficient and orthogonal bioconjugations in intact cells.


2015-16 Courses

Graduate and Fellowship Programs


All Publications

  • Organocatalytic removal of formaldehyde adducts from RNA and DNA bases NATURE CHEMISTRY Karmakar, S., Harcourt, E. M., Hewings, D. S., Lovejoy, A. F., Kurtz, D. M., Ehrenschwender, T., Barandun, L. J., Roost, C., Alizadeh, A. A., Kool, E. T. 2015; 7 (9): 752-758

    View details for DOI 10.1038/NCHEM.2307

    View details for Web of Science ID 000360191800014

  • In Vitro Fluorogenic Real-Time Assay of the Repair of Oxidative DNA Damage CHEMBIOCHEM Edwards, S. K., Ono, T., Wang, S., Jiang, W., Franzini, R. M., Jung, J. W., Chan, K. M., Kool, E. T. 2015; 16 (11): 1637-1646


    The repair of oxidative damage to DNA is essential to avoid mutations that lead to cancer. Oxidized DNA bases, such as 8-oxoguanine, are a main source of these mutations, and the enzyme 8-oxoguanine glycosylase 1 (OGG1) is the chief human enzyme that excises 8-oxoguanine from DNA. The activity of OGG1 has been linked to human inflammation responses and to cancer, and researchers are beginning to search for inhibitors of the enzyme. However, measuring the activity of the enzyme typically requires laborious gel-based measurements of radiolabeled DNAs. Here we report the design and properties of fluorogenic probes that directly report on the activity of OGG1 (and its bacterial homologue Fpg) in real time as the oxidized base is excised. The probes are short, modified DNA oligomers containing fluorescent DNA bases and are designed to utilize 8-oxoguanine itself as a fluorescence quencher. Screening of combinations of fluorophores and 8-oxoguanine revealed two fluorophores, pyrene and tCo, that are strongly quenched by the damaged base. We tested 42 potential probes containing these fluorophores: the optimum probe, OGR1, yields a 60-fold light-up signal in vitro with OGG1 and Fpg. It can report on oxidative repair activity in mammalian cell lysate and with bacterial cells overexpressing a repair enzyme. Such probes might prove useful in quantifying enzyme activity and performing competitive inhibition assays.

    View details for DOI 10.1002/cbic.201500184

    View details for Web of Science ID 000358330300015

    View details for PubMedID 26073452

  • Structural imprints in vivo decode RNA regulatory mechanisms. Nature Spitale, R. C., Flynn, R. A., Zhang, Q. C., Crisalli, P., Lee, B., Jung, J., Kuchelmeister, H. Y., Batista, P. J., Torre, E. A., Kool, E. T., Chang, H. Y. 2015; 519 (7544): 486-490


    Visualizing the physical basis for molecular behaviour inside living cells is a great challenge for biology. RNAs are central to biological regulation, and the ability of RNA to adopt specific structures intimately controls every step of the gene expression program. However, our understanding of physiological RNA structures is limited; current in vivo RNA structure profiles include only two of the four nucleotides that make up RNA. Here we present a novel biochemical approach, in vivo click selective 2'-hydroxyl acylation and profiling experiment (icSHAPE), which enables the first global view, to our knowledge, of RNA secondary structures in living cells for all four bases. icSHAPE of the mouse embryonic stem cell transcriptome versus purified RNA folded in vitro shows that the structural dynamics of RNA in the cellular environment distinguish different classes of RNAs and regulatory elements. Structural signatures at translational start sites and ribosome pause sites are conserved from in vitro conditions, suggesting that these RNA elements are programmed by sequence. In contrast, focal structural rearrangements in vivo reveal precise interfaces of RNA with RNA-binding proteins or RNA-modification sites that are consistent with atomic-resolution structural data. Such dynamic structural footprints enable accurate prediction of RNA-protein interactions and N(6)-methyladenosine (m(6)A) modification genome wide. These results open the door for structural genomics of RNA in living cells and reveal key physiological structures controlling gene expression.

    View details for DOI 10.1038/nature14263

    View details for PubMedID 25799993

  • Structural imprints in vivo decode RNA regulatory mechanisms NATURE Spitale, R. C., Flynn, R. A., Zhang, Q. C., Crisalli, P., Lee, B., Jung, J., Kuchelmeister, H. Y., Batista, P. J., Torre, E. A., Kool, E. T., Chang, H. Y. 2015; 519 (7544): 486-?
  • Structure and Thermodynamics of N-6-Methyladenosine in RNA: A Spring-Loaded Base Modification JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Roost, C., Lynch, S. R., Batista, P. J., Qu, K., Chang, H. Y., Kool, E. T. 2015; 137 (5): 2107-2115


    N(6)-Methyladenosine (m(6)A) modification is hypothesized to control processes such as RNA degradation, localization, and splicing. However, the molecular mechanisms by which this occurs are unclear. Here, we measured structures of an RNA duplex containing m(6)A in the GGACU consensus, along with an unmodified RNA control, by 2D NMR. The data show that m(6)A-U pairing in the double-stranded context is accompanied by the methylamino group rotating from its energetically preferred syn geometry on the Watson-Crick face to the higher-energy anti conformation, positioning the methyl group in the major groove. Thermodynamic measurements of m(6)A in duplexes reveal that it is destabilizing by 0.5-1.7 kcal/mol. In contrast, we show that m(6)A in unpaired positions base stacks considerably more strongly than the unmodified base, adding substantial stabilization in single-stranded locations. Transcriptome-wide nuclease mapping of methylated RNA secondary structure from human cells reveals a structural transition at methylated adenosines, with a tendency to single-stranded structure adjacent to the modified base.

    View details for DOI 10.1021/ja513080v

    View details for Web of Science ID 000349575800056

    View details for PubMedID 25611135

  • New Organocatalyst Scaffolds with High Activity in Promoting Hydrazone and Oxime Formation at Neutral pH ORGANIC LETTERS Larsen, D., Pittelkow, M., Karmakar, S., Kool, E. T. 2015; 17 (2): 274-277


    The discovery of two new classes of catalysts for hydrazone and oxime formation in water at neutral pH, namely 2-aminophenols and 2-(aminomethyl)benzimidazoles, is reported. Kinetics studies in aqueous solutions at pH 7.4 revealed rate enhancements up to 7-fold greater than with classic aniline catalysis. 2-(Aminomethyl)benzimidazoles were found to be effective catalysts with otherwise challenging aryl ketone substrates.

    View details for DOI 10.1021/ol503372j

    View details for Web of Science ID 000348331800027

    View details for PubMedID 25545888

  • Pattern-Based Detection of Anion Pollutants in Water with DNA Polyfluorophores CHEMICAL SCIENCE Kwon, H., Jei, W., Kool, E. T. 2015; 6: 2575-2583

    View details for DOI 10.1039/C4SC03992K

  • Pattern-based detection of anion pollutants in water with DNA polyfluorophores CHEMICAL SCIENCE Kwon, H., Jiang, W., Kool, E. T. 2015; 6 (4): 2575-2583

    View details for DOI 10.1039/c4sc03992k

    View details for Web of Science ID 000351412800054

  • Structure and Thermodynamics of N6-Methyladenosine in RNA: A Spring-Loaded Base Modification Journal of American Chemical Society Roost, C., Lynch, S. M., Batista, P. J., Qu, K., Chang, H. Y., Kool, E. T. 2015; 137: 2107-2115
  • RNA structural analysis by evolving SHAPE chemistry WILEY INTERDISCIPLINARY REVIEWS-RNA Spitale, R. C., Flynn, R. A., Torre, E. A., Kool, E. T., Chang, H. Y. 2014; 5 (6): 867-881

    View details for DOI 10.1002/wrna.1253

    View details for Web of Science ID 000344453000009

  • Large-Scale Detection of Metals with a Small Set of Fluorescent DNA-Like Chemosensors JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Yuen, L. H., Franzini, R. M., Tan, S. S., Kool, E. T. 2014; 136 (41): 14576-14582


    An important advantage of pattern-based chemosensor sets is their potential to detect and differentiate a large number of analytes with only few sensors. Here we test this principle at a conceptual limit by analyzing a large set of metal ion analytes covering essentially the entire periodic table, employing fluorescent DNA-like chemosensors on solid support. A tetrameric "oligodeoxyfluoroside" (ODF) library of 6561 members containing metal-binding monomers was screened for strong responders to 57 metal ions in solution. Our results show that a set of 9 chemosensors could successfully discriminate the 57 species, including alkali, alkaline earth, post-transition, transition, and lanthanide metals. As few as 6 ODF chemosensors could detect and differentiate 50 metals at 100 μM; sensitivity for some metals was achieved at midnanomolar ranges. A blind test with 50 metals further confirmed the discriminating power of the ODFs.

    View details for DOI 10.1021/ja507932a

    View details for Web of Science ID 000343276900042

    View details for PubMedID 25255102

  • Molecular basis of transcriptional fidelity and DNA lesion-induced transcriptional mutagenesis. DNA repair Xu, L., Da, L., Plouffe, S. W., Chong, J., Kool, E., Wang, D. 2014; 19: 71-83


    Maintaining high transcriptional fidelity is essential for life. Some DNA lesions lead to significant changes in transcriptional fidelity. In this review, we will summarize recent progress towards understanding the molecular basis of RNA polymerase II (Pol II) transcriptional fidelity and DNA lesion-induced transcriptional mutagenesis. In particular, we will focus on the three key checkpoint steps of controlling Pol II transcriptional fidelity: insertion (specific nucleotide selection and incorporation), extension (differentiation of RNA transcript extension of a matched over mismatched 3'-RNA terminus), and proofreading (preferential removal of misincorporated nucleotides from the 3'-RNA end). We will also discuss some novel insights into the molecular basis and chemical perspectives of controlling Pol II transcriptional fidelity through structural, computational, and chemical biology approaches.

    View details for DOI 10.1016/j.dnarep.2014.03.024

    View details for PubMedID 24767259

  • Molecular basis of transcriptional fidelity and DNA lesion-induced transcriptional mutagenesis DNA REPAIR Xu, L., Da, L., Plouffe, S. W., Chong, J., Kool, E., Wang, D. 2014; 19: 71-83
  • Pattern-Based Detection of Toxic Metals in Surface Water with DNA Polyfluorophores** ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Yuen, L. H., Franzini, R. M., Wang, S., Crisalli, P., Singh, V., Jiang, W., Kool, E. T. 2014; 53 (21): 5361-5365


    Heavy metal contamination of water can be toxic to humans and wildlife; thus the development of methods to detect this contamination is of high importance. Here we describe the design and application of DNA-based fluorescent chemosensors on microbeads to differentiate eight toxic metal ions in water. We developed and synthesized four fluorescent 2'-deoxyribosides of metal-binding ligands. A tetramer-length oligodeoxy-fluoroside (ODF) library of 6561 members was constructed and screened for sequences responsive to metal ions, of which seven sequences were selected. Statistical analysis of the response patterns showed successful differentiation of the analytes at concentrations as low as 100 nM. Sensors were able to classify water samples from 13 varied sites and quantify metal contamination in unknown specimens. The results demonstrate the practical potential of bead-based ODF chemosensors to analyze heavy metal contamination in water samples by a simple and inexpensive optical method.

    View details for DOI 10.1002/anie.201403235

    View details for Web of Science ID 000335809200019

  • Fast Alpha Nucleophiles: Structures that Undergo Rapid Hydrazone/Oxime Formation at Neutral pH ORGANIC LETTERS Kool, E. T., Crisalli, P., Chan, K. M. 2014; 16 (5): 1454-1457


    Hydrazones and oximes are widely useful structures for conjugate formation in chemistry and biology, but their formation can be slow at neutral pH. Kinetics studies were performed for a range of structurally varied hydrazines, and a surprisingly large variation in reaction rate was observed. Structures that undergo especially rapid reactions were identified, enabling reaction rates that rival orthogonal cycloaddition-based conjugation chemistries.

    View details for DOI 10.1021/ol500262y

    View details for Web of Science ID 000332756400046

    View details for PubMedID 24559274

  • Designer bases, base pairs, and genetic sets: biochemical and biological activity, in Synthetic Biology: Volume 1, 2014, 1, pp. 1-30 Synthetic Biology Hartcourt, E. M., Kool, E. T. Royal Society of Chemistry. 2014: 1-30
  • Dissecting the chemical interactions and substrate structural signatures governing RNA polymerase II trigger loop closure by synthetic nucleic acid analogues NUCLEIC ACIDS RESEARCH Xu, L., Butler, K. V., Chong, J., Wengel, J., Kool, E. T., Wang, D. 2014; 42 (9): 5863-5870


    The trigger loop (TL) of RNA polymerase II (Pol II) is a conserved structural motif that is crucial for Pol II catalytic activity and transcriptional fidelity. The TL remains in an inactive open conformation when the mismatched substrate is bound. In contrast, TL switches from an inactive open state to a closed active state to facilitate nucleotide addition upon the binding of the cognate substrate to the Pol II active site. However, a comprehensive understanding of the specific chemical interactions and substrate structural signatures that are essential to this TL conformational change remains elusive. Here we employed synthetic nucleotide analogues as 'chemical mutation' tools coupling with α-amanitin transcription inhibition assay to systematically dissect the key chemical interactions and structural signatures governing the substrate-coupled TL closure in Saccharomyces cerevisiae Pol II. This study reveals novel insights into understanding the molecular basis of TL conformational transition upon substrate binding during Pol II transcription. This synthetic chemical biology approach may be extended to understand the mechanisms of other RNA polymerases as well as other nucleic acid enzymes in future studies.

    View details for DOI 10.1093/nar/gku238

    View details for Web of Science ID 000336495400046

  • Large-scale Detection of Metals with a Small Number of DNA-like Fluorescent Chemosensors Journal of the American Chemical Society Yuen, L. H., Franzini, R. M., Tan, S. S., Kool, E. T. 2014
  • Identification of a Selective Polymerase Enables Detection of N-6-Methyladenosine in RNA JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Harcourt, E. M., Ehrenschwender, T., Batista, P. J., Chang, H. Y., Kool, E. T. 2013; 135 (51): 19079-19082


    N(6)-methyladenosine (m(6)A) is the most abundant mRNA modification and has important links to human health. While recent studies have successfully identified thousands of mammalian RNA transcripts containing the modification, it is extremely difficult to identify the exact location of any specific m(6)A. Here we have identified a polymerase with reverse transcriptase activity (from Thermus thermophilus) that is selective by up to 18-fold for incorporation of thymidine opposite unmodified A over m(6)A. We show that the enzyme can be used to locate and quantify m(6)A in synthetic RNAs by analysis of pausing bands, and have used the enzyme in tandem with a nonselective polymerase to locate the presence and position of m(6)A in high-abundance cellular RNAs. By this approach we demonstrate that the long-undetermined position of m(6)A in mammalian 28S rRNA is nucleotide 4190.

    View details for DOI 10.1021/ja4105792

    View details for Web of Science ID 000329137300009

    View details for PubMedID 24328136

  • Fast Hydrazone Reactants: Electronic and Acid/Base Effects Strongly Influence Rate at Biological pH JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Kool, E. T., Park, D., Crisalli, P. 2013; 135 (47): 17663-17666


    Kinetics studies with structurally varied aldehydes and ketones in aqueous buffer at pH 7.4 reveal that carbonyl compounds with neighboring acid/base groups form hydrazones at accelerated rates. Similarly, tests of a hydrazine with a neighboring carboxylic acid group show that it also reacts at an accelerated rate. Rate constants for the fastest carbonyl/hydrazine combinations are 2-20 M(-1) s(-1), which is faster than recent strain-promoted cycloaddition reactions.

    View details for DOI 10.1021/ja407407h

    View details for Web of Science ID 000328099400003

    View details for PubMedID 24224646

  • Artificial Genetic Sets Composed of Size-Expanded Base Pairs ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Winnacker, M., Kool, E. T. 2013; 52 (48): 12498-12508


    We describe in this Minireview the synthesis, properties, and applications of artificial genetic sets built from base pairs that are larger than the natural Watson-Crick architecture. Such designed systems are being explored by several research groups to investigate basic chemical questions regarding the functions of the genetic information storage systems and thus of the origin and evolution of life. For example, is the terrestrial DNA structure the only viable one, or can other architectures function as well? Working outside the constraints of purine-pyrimidine geometry provides more chemical flexibility in design, and the added size confers useful properties such as high binding affinity and helix stability as well as fluorescence. These features are useful for the investigation of fundamental biochemical questions as well as in the development of new biotechnological, biomedical, and nanostructural tools and methods.

    View details for DOI 10.1002/anie.201305267

    View details for Web of Science ID 000327582900005

    View details for PubMedID 24249550

  • Monitoring eukaryotic and bacterial UDG repair activity with DNA-multifluorophore sensors. Nucleic acids research Ono, T., Edwards, S. K., Wang, S., Jiang, W., Kool, E. T. 2013; 41 (12)


    We report the development of simple fluorogenic probes that report on the activity of both bacterial and mammalian uracil-DNA glycosylase (UDG) enzymes. The probes are built from short, modified single-stranded oligonucleotides containing natural and unnatural bases. The combination of multiple fluorescent pyrene and/or quinacridone nucleobases yields fluorescence at 480 and 540 nm (excitation 340 nm), with large Stokes shifts of 140-200 nm, considerably greater than previous probes. They are strongly quenched by uracil bases incorporated into the sequence, and they yield light-up signals of up to 40-fold, or ratiometric signals with ratio changes of 82-fold, on enzymatic removal of these quenching uracils. We find that the probes are efficient reporters of bacterial UDG, human UNG2, and human SMUG1 enzymes in vitro, yielding complete signals in minutes. Further experiments establish that a probe can be used to image UDG activity by laser confocal microscopy in bacterial cells and in a human cell line, and that signals from a probe signalling UDG activity in human cells can be quantified by flow cytometry. Such probes may prove generally useful both in basic studies of these enzymes and in biomedical applications as well.

    View details for DOI 10.1093/nar/gkt309

    View details for PubMedID 23644286

  • Monitoring eukaryotic and bacterial UDG repair activity with DNA-multifluorophore sensors NUCLEIC ACIDS RESEARCH Ono, T., Edwards, S. K., Wang, S., Jiang, W., Kool, E. T. 2013; 41 (12)

    View details for DOI 10.1093/nar/gkt309

    View details for Web of Science ID 000321057100008

  • Genetically encoded multispectral labeling of proteins with polyfluorophores on a DNA backbone. Journal of the American Chemical Society Singh, V., Wang, S., Kool, E. T. 2013; 135 (16): 6184-6191


    Genetically encoded methods for protein conjugation are of high importance as biological tools. Here we describe the development of a new class of dyes for genetically encoded tagging that add new capabilities for protein reporting and detection via HaloTag methodology. Oligodeoxyfluorosides (ODFs) are short DNA-like oligomers in which the natural nucleic acid bases are replaced by interacting fluorescent chromophores, yielding a broad range of emission colors using a single excitation wavelength. We describe the development of an alkyl halide dehalogenase-compatible chloroalkane linker phosphoramidite derivative that enables the rapid automated synthesis of many possible dyes for protein conjugation. Experiments to test the enzymatic self-conjugation of nine different DNA-like dyes to proteins with HaloTag domains in vitro were performed, and the data confirmed the rapid and efficient covalent labeling of the proteins. Notably, a number of the ODF dyes were found to increase in brightness or change color upon protein conjugation. Tests in mammalian cellular settings revealed that the dyes are functional in multiple cellular contexts, both on the cell surface and within the cytoplasm, allowing protein localization to be imaged in live cells by epifluorescence and laser confocal microscopy.

    View details for DOI 10.1021/ja4004393

    View details for PubMedID 23590213

  • Importance of ortho Proton Donors in Catalysis of Hydrazone Formation ORGANIC LETTERS Crisalli, P., Kool, E. T. 2013; 15 (7): 1646-1649


    Anthranilic acids were recently reported as superior catalysts for hydrazone and oxime formation compared to aniline, the classic catalyst for these reactions. Here, alternative proton donors were examined with varied pKa in an effort to enhance activity at biological pH. The experiments show that 2-aminobenzenephosphonic acids are superior to anthranilic acids in catalyzing hydrazone formation with common aldehyde substrates.

    View details for DOI 10.1021/ol400427x

    View details for Web of Science ID 000317327900058

    View details for PubMedID 23477719

  • Selective Fluorogenic Chemosensors for Distinct Classes of Nucleases CHEMBIOCHEM Jung, J., Edwards, S. K., Kool, E. T. 2013; 14 (4): 440-444


    NUCLEASE SENSOR TRIO: Fluorogenic DNA sensors were developed for distinct classes of nucleases: 3'-exonucleases, 5'-exonucleases, and endonucleases. The highly selective sensors, built from very small modified DNA oligomers containing the unnatural fluorescent base pyrene, and employing thymine as a quencher, were found to function in a variety of complex biological media.

    View details for DOI 10.1002/cbic.201300001

    View details for Web of Science ID 000315492200006

    View details for PubMedID 23371801

  • Water-Soluble Organocatalysts for Hydrazone and Oxime Formation JOURNAL OF ORGANIC CHEMISTRY Crisalli, P., Kool, E. T. 2013; 78 (3): 1184-1189


    The formation of oximes and hydrazones is widely used in chemistry and biology as a molecular conjugation strategy for achieving ligation, attachment, and bioconjugation. However, the relatively slow rate of reaction has hindered its utility. Here, we report that simple, commercially available anthranilic acids and aminobenzoic acids act as superior catalysts for hydrazone and oxime formation, speeding the reaction considerably over the traditional aniline-catalyzed reaction at neutral pH. This efficient nucleophilic catalysis, involving catalyst-imine intermediates, allows rapid hydrazone/oxime formation even with relatively low concentrations of the two reactants. The most efficient catalysts are found to be 5-methoxyanthranilic acid and 3,5-diaminobenzoic acid; we find that they can enhance rates by factors of as much as 1-2 orders of magnitude over the aniline-catalyzed reaction. Evidence based on a range of differently substituted arylamines suggests that the ortho-carboxylate group in the anthranilate catalysts serves to aid in intramolecular proton transfer during imine and hydrazone formation.

    View details for DOI 10.1021/jo302746p

    View details for Web of Science ID 000314558300039

    View details for PubMedID 23289546

  • DNA-polyfluorophore chemosensors for environmental remediation: vapor-phase identification of petroleum products in contaminated soil CHEMICAL SCIENCE Jiang, W., Wang, S., Yuen, L. H., Kwon, H., Ono, T., Kool, E. T. 2013; 4 (8): 3184-3190


    Contamination of soil and groundwater by petroleum-based products is an extremely widespread and important environmental problem. Here we have tested a simple optical approach for detecting and identifying such industrial contaminants in soil samples, using a set of fluorescent DNA-based chemosensors in pattern-based sensing. We used a set of diverse industrial volatile chemicals to screen and identify a set of five short oligomeric DNA fluorophores on PEG-polystyrene microbeads that could differentiate the entire set after exposure to their vapors in air. We then tested this set of five fluorescent chemosensor compounds for their ability to respond with fluorescence changes when exposed to headgas over soil samples contaminated with one of ten different samples of crude oil, petroleum distillates, fuels, lubricants and additives. Statistical analysis of the quantitative fluorescence change data (as Δ(R,G,B) emission intensities) revealed that these five chemosensors on beads could differentiate all ten product mixtures at 1000 ppm in soil within 30 minutes. Tests of sensitivity with three of the contaminant mixtures showed that they could be detected and differentiated in amounts at least as low as one part per million in soil. The results establish that DNA-polyfluorophores may have practical utility in monitoring the extent and identity of environmental spills and leaks, while they occur and during their remediation.

    View details for DOI 10.1039/c3sc50985k

    View details for Web of Science ID 000321307000025

  • Chemical fidelity of an RNA polymerase ribozyme CHEMICAL SCIENCE Attwater, J., Tagami, S., Kimoto, M., Butler, K., Kool, E. T., Wengel, J., Herdewijn, P., Hirao, I., Holliger, P. 2013; 4 (7): 2804-2814

    View details for DOI 10.1039/c3sc50574j

    View details for Web of Science ID 000319940400011

  • RNA SHAPE analysis in living cells NATURE CHEMICAL BIOLOGY Spitale, R. C., Crisalli, P., Flynn, R. A., Torre, E. A., Kool, E. T., Chang, H. Y. 2013; 9 (1): 18-?
  • RNA SHAPE analysis in living cells. Nature chemical biology Spitale, R. C., Crisalli, P., Flynn, R. A., Torre, E. A., Kool, E. T., Chang, H. Y. 2013; 9 (1): 18-20


    RNA structure has important roles in practically every facet of gene regulation, but the paucity of in vivo structural probes limits current understanding. Here we design, synthesize and demonstrate two new chemical probes that enable selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) in living cells. RNA structures in human, mouse, fly, yeast and bacterial cells are read out at single-nucleotide resolution, revealing tertiary contacts and RNA-protein interactions.

    View details for DOI 10.1038/nchembio.1131

    View details for PubMedID 23178934

  • Fluorescence Quenchers for Hydrazone and Oxime Orthogonal Bioconjugation BIOCONJUGATE CHEMISTRY Crisalli, P., Hernandez, A. R., Kool, E. T. 2012; 23 (9): 1969-1980


    We describe the synthesis and properties of new fluorescence quenchers containing aldehyde, hydrazine, and aminooxy groups, allowing convenient bioconjugation as oximes or hydrazones. Conjugation to oligonucleotides proceeded in high yield with aniline as catalyst. Kinetics studies of conjugation show that, under optimal conditions, a hydrazine or aminooxy quencher can react with aldehyde-modified DNA to form a stable hydrazone or oxime adduct in as little as five minutes. The resulting quencher-containing DNAs were assessed for their ability to quench the emission of fluorescein in labeled complements and compared to the commercially available dabcyl and Black Hole Quencher 2 (BHQ2), which were conjugated as phosphoramidites. Results show that the new quenchers possess slightly different absorbance properties compared to dabcyl and are as efficient as the commercial quenchers in quenching fluorescein emission. Hydrazone-based quenchers were further successfully incorporated into molecular beacons and shown to give high signal to background ratios in single nucleotide polymorphism detection in vitro. Finally, aminooxy and hydrazine quenchers were applied to quenching of an aldehyde-containing fluorophore associated with living cells, demonstrating cellular quenching within one hour.

    View details for DOI 10.1021/bc300344b

    View details for Web of Science ID 000308833600028

    View details for PubMedID 22913527

  • Steric Restrictions of RISC in RNA Interference Identified with Size-Expanded RNA Nucleobases ACS CHEMICAL BIOLOGY Hernandez, A. R., Peterson, L. W., Kool, E. T. 2012; 7 (8): 1454-1461


    Understanding the interactions between small interfering RNAs (siRNAs) and the RNA-induced silencing complex (RISC), the key protein complex of RNA interference (RNAi), is of great importance to the development of siRNAs with improved biological and potentially therapeutic function. Although various chemically modified siRNAs have been reported, relatively few studies with modified nucleobases exist. Here we describe the synthesis and hybridization properties of siRNAs bearing size-expanded RNA (xRNA) nucleobases and their use as a novel and systematic set of steric probes in RNAi. xRNA nucleobases are expanded by 2.4 Å using benzo-homologation and retain canonical Watson-Crick base-pairing groups. Our data show that the modified siRNA duplexes display small changes in melting temperature (+1.4 to -5.0 °C); substitutions near the center are somewhat destabilizing to the RNA duplex, while substitutions near the ends are stabilizing. RNAi studies in a dual-reporter luciferase assay in HeLa cells revealed that xRNA nucleobases in the antisense strand reduce activity at some central positions near the seed region but are generally well tolerated near the ends. Most importantly, we observed that xRNA substitutions near the 3'-end increased activity over that of wild-type siRNAs. The data are analyzed in terms of site-dependent steric effects in RISC. Circular dichroism experiments show that single xRNA substitutions do not significantly distort the native A-form helical structure of the siRNA duplex, and serum stability studies demonstrated that xRNA substitutions protect siRNAs against nuclease degradation.

    View details for DOI 10.1021/cb300174c

    View details for Web of Science ID 000307526600019

    View details for PubMedID 22646660

  • DNA-Multichromophore Systems CHEMICAL REVIEWS Teo, Y. N., Kool, E. T. 2012; 112 (7): 4221-4245

    View details for DOI 10.1021/cr100351g

    View details for Web of Science ID 000306298800015

    View details for PubMedID 22424059

  • Dissecting Chemical Interactions Governing RNA Polymerase II Transcriptional Fidelity JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Kellinger, M. W., Ulrich, S., Chong, J., Kool, E. T., Wang, D. 2012; 134 (19): 8231-8240


    Maintaining high transcriptional fidelity is essential to life. For all eukaryotic organisms, RNA polymerase II (Pol II) is responsible for messenger RNA synthesis from the DNA template. Three key checkpoint steps are important in controlling Pol II transcriptional fidelity: nucleotide selection and incorporation, RNA transcript extension, and proofreading. Some types of DNA damage significantly reduce transcriptional fidelity. However, the chemical interactions governing each individual checkpoint step of Pol II transcriptional fidelity and the molecular basis of how subtle DNA base damage leads to significant losses of transcriptional fidelity are not fully understood. Here we use a series of "hydrogen bond deficient" nucleoside analogues to dissect chemical interactions governing Pol II transcriptional fidelity. We find that whereas hydrogen bonds between a Watson-Crick base pair of template DNA and incoming NTP are critical for efficient incorporation, they are not required for efficient transcript extension from this matched 3'-RNA end. In sharp contrast, the fidelity of extension is strongly dependent on the discrimination of an incorrect pattern of hydrogen bonds. We show that U:T wobble base interactions are critical to prevent extension of this mismatch by Pol II. Additionally, both hydrogen bonding and base stacking play important roles in controlling Pol II proofreading activity. Strong base stacking at the 3'-RNA terminus can compensate for loss of hydrogen bonds. Finally, we show that Pol II can distinguish very subtle size differences in template bases. The current work provides the first systematic evaluation of electrostatic and steric effects in controlling Pol II transcriptional fidelity.

    View details for DOI 10.1021/ja302077d

    View details for Web of Science ID 000304027100039

    View details for PubMedID 22509745

  • Amplified microRNA detection by templated chemistry NUCLEIC ACIDS RESEARCH Harcourt, E. M., Kool, E. T. 2012; 40 (9)


    MicroRNAs (miRNAs) are a class of RNAs that play important regulatory roles in the cell. The detection of microRNA has attracted significant interest recently, as abnormal miRNA expression has been linked to cancer and other diseases. Here, we present a straightforward method for isothermal amplified detection of miRNA that involves two separate nucleic acid-templated chemistry steps. The miRNA first templates the cyclization of an oligodeoxynucleotide from a linear precursor containing a 5'-iodide and a 3'-phosphorothioate. The sequence is amplified through rolling circle amplification with 29 DNA polymerase and then detected via a second amplification using fluorogenic templated probes. Tests showed that the cyclization proceeds in ?50% yield over 24 h and is compatible with the conditions required for rolling circle polymerization, unlike enzymatic ligations which required non-compatible buffer conditions. The polymerization yielded 188-fold amplification, and separate experiments showed ?15-fold signal amplification from the templated fluorogenic probes. When all components are combined, results show miRNA detection down to 200 pM in solution, and correlation of the detected signal with the initial concentration of miRNA. The doubly templated double-amplification method demonstrates a new approach to detection of rolling circle products and significant advantages in ease of operation for miRNA detection.

    View details for DOI 10.1093/nar/gkr1313

    View details for Web of Science ID 000304201300003

    View details for PubMedID 22278881

  • Nonpolar nucleosides alter RNA Polymerase II NTP specificity by disrupting hydrogen bonding and base stacking Kellinger, M. W., Ulrich, S., Kool, E. T., Wang, D. FEDERATION AMER SOC EXP BIOL. 2012
  • Measurement and Theory of Hydrogen Bonding Contribution to Isosteric DNA Base Pairs JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Khakshoor, O., Wheeler, S. E., Houk, K. N., Kool, E. T. 2012; 134 (6): 3154-3163


    We address the recent debate surrounding the ability of 2,4-difluorotoluene (F), a low-polarity mimic of thymine (T), to form a hydrogen-bonded complex with adenine in DNA. The hydrogen bonding ability of F has been characterized as small to zero in various experimental studies, and moderate to small in computational studies. However, recent X-ray crystallographic studies of difluorotoluene in DNA/RNA have indicated, based on interatomic distances, possible hydrogen bonding interactions between F and natural bases in nucleic acid duplexes and in a DNA polymerase active site. Since F is widely used to measure electrostatic contributions to pairing and replication, it is important to quantify the impact of this isostere on DNA stability. Here, we studied the pairing stability and selectivity of this compound and a closely related variant, dichlorotoluene deoxyriboside (L), in DNA, using both experimental and computational approaches. We measured the thermodynamics of duplex formation in three sequence contexts and with all possible pairing partners by thermal melting studies using the van't Hoff approach, and for selected cases by isothermal titration calorimetry (ITC). Experimental results showed that internal F-A pairing in DNA is destabilizing by 3.8 kcal/mol (van't Hoff, 37 °C) as compared with T-A pairing. At the end of a duplex, base-base interactions are considerably smaller; however, the net F-A interaction remains repulsive while T-A pairing is attractive. As for selectivity, F is found to be slightly selective for adenine over C, G, T by 0.5 kcal mol, as compared with thymine's selectivity of 2.4 kcal/mol. Interestingly, dichlorotoluene in DNA is slightly less destabilizing and slightly more selective than F, despite the lack of strongly electronegative fluorine atoms. Experimental data were complemented by computational results, evaluated at the M06-2X/6-31+G(d) and MP2/cc-pVTZ levels of theory. These computations suggest that the pairing energy of F to A is ~28% of that of T-A, and most of this interaction does not arise from the F···HN interaction, but rather from the CH···N interaction. The nucleobase analogue shows no inherent selectivity for adenine over other bases, and L-A pairing energies are slightly weaker than for F-A. Overall, the results are consistent with a small favorable noncovalent interaction of F with A offset by a large desolvation cost for the polar partner. We discuss the findings in light of recent structural studies and of DNA replication experiments involving these analogues.

    View details for DOI 10.1021/ja210475a

    View details for Web of Science ID 000301161500056

    View details for PubMedID 22300089

  • Surprising Repair Activities of Nonpolar Analogs of 8-oxoG Expose Features of Recognition and Catalysis by BaSe Excision Repair Glycosylases JOURNAL OF THE AMERICAN CHEMICAL SOCIETY McKibbin, P. L., Kobori, A., Taniguchi, Y., Kool, E. T., David, S. S. 2012; 134 (3): 1653-1661


    Repair glycosylases locate and excise damaged bases from DNA, playing central roles in preservation of the genome and prevention of disease. Two key glycosylases, Fpg and hOGG1, function to remove the mutagenic oxidized base 8-oxoG (OG) from DNA. To investigate the relative contributions of conformational preferences, leaving group ability, enzyme-base hydrogen bonding, and nucleobase shape on damage recognition by these glycosylases, a series of four substituted indole nucleosides, based on the parent OG nonpolar isostere 2Cl-4F-indole, were tested as possible direct substrates of these enzymes in the context of 30 base pair duplexes paired with C. Surprisingly, single-turnover experiments revealed that Fpg-catalyzed base removal activity of two of the nonpolar analogs was superior to the native OG substrate. The hOGG1 glycosylase was also found to catalyze removal of three of the nonpolar analogs, albeit considerably less efficiently than removal of OG. Of note, the analog that was completely resistant to hOGG1-catalyzed excision has a chloro-substituent at the position of NH7 of OG, implicating the importance of recognition of this position in catalysis. Both hOGG1 and Fpg retained high affinity for the duplexes containing the nonpolar isosteres. These studies show that hydrogen bonds between base and enzyme are not needed for efficient damage recognition and repair by Fpg and underscore the importance of facile extrusion from the helix in its damaged base selection. In contrast, damage removal by hOGG1 is sensitive to both hydrogen bonding groups and nucleobase shape. The relative rates of excision of the analogs with the two glycosylases highlight key differences in their mechanisms of damaged base recognition and removal.

    View details for DOI 10.1021/ja208510m

    View details for Web of Science ID 000301084400051

    View details for PubMedID 22175854

  • Templated chemistry for monitoring damage and repair directly in duplex DNA CHEMICAL COMMUNICATIONS Lee, S. H., Wang, S., Kool, E. T. 2012; 48 (65): 8069-8071


    We report the fluorogenic detection of the product of base excision repair (an abasic site) in a specific sequence of duplex DNA. This is achieved by DNA-templated chemistry, employing triple helix-forming probes that contain unnatural nucleobases designed to selectively recognize the site of a missing base. Light-up signals of up to 36-fold were documented, and probes could be used to monitor enzymatic removal of a damaged base.

    View details for DOI 10.1039/c2cc34060g

    View details for Web of Science ID 000306573800008

    View details for PubMedID 22782065

  • Fluorescent DNAs printed on paper: sensing food spoilage and ripening in the vapor phase CHEMICAL SCIENCE Kwon, H., Samain, F., Kool, E. T. 2012; 3 (8): 2542-2549

    View details for DOI 10.1039/c2sc20461d

    View details for Web of Science ID 000305900700018

  • DNA Polyfluorophores for Real-Time Multicolor Tracking of Dynamic Biological Systems ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Wang, S., Guo, J., Ono, T., Kool, E. T. 2012; 51 (29): 7176-7180


    Dye-ing to live: Spectral limitations of common organic dyes make it difficult or impossible to visualize and follow multiple biological components in rapidly moving systems. The development of a multispectral set of improved DNA-scaffolded fluorophores is described. Their use in multicolor cellular imaging (see scheme) and in tracking of biological motions on the subsecond timescale is demonstrated.

    View details for DOI 10.1002/anie.201201928

    View details for Web of Science ID 000306314300020

    View details for PubMedID 22684777

  • Direct Fluorescence Monitoring of DNA Base Excision Repair ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Ono, T., Wang, S., Koo, C., Engstrom, L., David, S. S., Kool, E. T. 2012; 51 (7): 1689-1692

    View details for DOI 10.1002/anie.201108135

    View details for Web of Science ID 000299946400036

    View details for PubMedID 22241823

  • Importance of Steric Effects on the Efficiency and Fidelity of Transcription by T7 RNA Polymerase BIOCHEMISTRY Ulrich, S., Kool, E. T. 2011; 50 (47): 10343-10349


    DNA-dependent RNA polymerases such as T7 RNA polymerase (T7 RNAP) perform the transcription of DNA into mRNA with high efficiency and high fidelity. Although structural studies have provided a detailed account of the molecular basis of transcription, the relative importance of factors like hydrogen bonds and steric effects remains poorly understood. We report herein the first study aimed at systematically probing the importance of steric and electrostatic effects on the efficiency and fidelity of DNA transcription by T7 RNAP. We used synthetic nonpolar analogues of thymine with sizes varying in subangstrom increments to probe the steric requirements of T7 RNAP during the elongation mode of transcription. Enzymatic assays with internal radiolabeling were performed to compare the efficiency of transcription of modified DNA templates with a natural template containing thymine as a reference. Furthermore, we analyzed effects on the fidelity by measuring the composition of RNA transcripts by enzymatic digestion followed by two-dimensional thin layer chromatography separation. Our results demonstrate that hydrogen bonds play an important role in the efficiency of transcription but, interestingly, do not appear to be required for faithful transcription. Steric effects (size and shape variations) are found to be significant both in insertion of a new RNA base and in extension beyond it.

    View details for DOI 10.1021/bi2011465

    View details for Web of Science ID 000297143700018

    View details for PubMedID 22044042

  • Encoding Phenotype in Bacteria with an Alternative Genetic Set JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Krueger, A. T., Peterson, L. W., Chelliserry, J., Kleinbaum, D. J., Kool, E. T. 2011; 133 (45): 18447-18451


    An unnatural base-pair architecture with base pairs 2.4 Å larger than the natural DNA-based genetic system (xDNA) is evaluated for its ability to function like DNA, encoding amino acids in the context of living cells. xDNA bases are structurally analogous to natural bases but homologated by the width of a benzene ring, increasing their sizes and resulting in a duplex that is wider than native B-DNA. Plasmids encoding green fluorescent protein were constructed to contain single and multiple xDNA bases (as many as eight) in both strands and were transformed into Escherichia coli. Although they yielded fewer colonies than the natural control plasmid, in all cases in which a modified plasmid (containing one, two, three, or four consecutive size-expanded base pairs) was used, the correct codon bases were substituted, yielding green colonies. All four xDNA bases (xA, xC, xG, and xT) were found to encode the correct partners in the replicated plasmid DNA, both alone and in longer segments of xDNA. Controls with mutant cell lines having repair functions deleted were found to express the gene correctly, ruling out repair of xDNA and confirming polymerase reading of the unnatural bases. Preliminary experiments with polymerase deletion mutants suggested combined roles of replicative and lesion-bypass polymerases in inserting correct bases opposite xDNA bases and in bypassing the xDNA segments. These experiments demonstrate a biologically functioning synthetic genetic set with larger-than-natural architecture.

    View details for DOI 10.1021/ja208025e

    View details for Web of Science ID 000297381200070

    View details for PubMedID 21981660

  • Multi-Path Quenchers: Efficient Quenching of Common Fluorophores BIOCONJUGATE CHEMISTRY Crisalli, P., Kool, E. T. 2011; 22 (11): 2345-2354


    Fluorescence quenching groups are widely employed in biological detection, sensing, and imaging. To date, a relatively small number of such groups are in common use. Perhaps the most commonly used quencher, dabcyl, has limited efficiency with a broad range of fluorophores. Here, we describe a molecular approach to improve the efficiency of quenchers by increasing their electronic complexity. Multi-Path Quenchers (MPQ) are designed to have multiple donor or acceptor groups in their structure, allowing for a multiplicity of conjugation pathways of varied length. This has the effect of broadening the absorption spectrum, which in turn can increase quenching efficiency and versatility. Six such MPQ derivatives are synthesized and tested for quenching efficiency in a DNA hybridization context. Duplexes placing quenchers and fluorophores within contact distance or beyond this distance are used to measure quenching via contact or FRET mechanisms. Results show that several of the quenchers are considerably more efficient than dabcyl at quenching a wider range of common fluorophores, and two quench fluorescein and TAMRA as well as or better than a Black Hole Quencher.

    View details for DOI 10.1021/bc200424r

    View details for Web of Science ID 000297001800018

    View details for PubMedID 22034828

  • Improved Templated Fluorogenic Probes Enhance the Analysis of Closely Related Pathogenic Bacteria by Microscopy and Flow Cytometry BIOCONJUGATE CHEMISTRY Franzini, R. M., Kool, E. T. 2011; 22 (9): 1869-1877


    Templated fluorescence activation has recently emerged as a promising molecular approach to detect and differentiate nucleic acid sequences in vitro and in cells. Here, we describe the application of a reductive quencher release strategy to the taxonomic analysis of Gram-negative bacteria by targeting a single nucleotide difference in their 16S rRNA in a two-color assay. For this purpose, it was necessary to develop a release linker containing a quencher suitable for red and near-infrared fluorophores, and to improve methods for the delivery of probes into cells. A cyanine-dye labeled oligonucleotide probe containing the new quencher-release linker showed unprecedentedly low background signal and high fluorescence turn-on ratios. The combination of a fluorescein-containing and a near-IR emitting probe discriminated E. coli from S. enterica despite nearly identical ribosomal target sequences. Two-color analysis by microscopy and the first successful discrimination of bacteria by two-color flow cytometry with templated reactive probes are described.

    View details for DOI 10.1021/bc2003567

    View details for Web of Science ID 000295059000016

    View details for PubMedID 21870777

  • Polyfluorophores on a DNA backbone: Expanded spectrum and improved stability with new fluorophores Wang, S., Guo, J., Kool, E. T. AMER CHEMICAL SOC. 2011
  • Differentiating between Fluorescence-Quenching Metal Ions with Polyfluorophore Sensors Built on a DNA Backbone JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Tan, S. S., Kim, S. J., Kool, E. T. 2011; 133 (8): 2664-2671


    A common problem in detecting metal ions with fluorescentchemosensors is the emission-suppressing effects of fluorescence-quenching metal ions. This quenching tendency makes it difficult to design sensors with turn-on signal, and differentiate between several metal ions that may yield a strong quenching response. To address these challenges, we investigate a new sensor design strategy, incorporating fluorophores and metal ligands as DNA base replacements in DNA-like oligomers, for generating a broader range of responses for quenching metal ions. The modular molecular design enabled rapid synthesis and discovery of sensors from libraries on PEG-polystyrene beads. Using this approach, water-soluble sensors 1-5 were identified as strong responders to a set of eight typically quenching metal ions (Co(2+), Ni(2+), Cu(2+), Hg(2+), Pb(2+), Ag(+), Cr(3+), and Fe(3+)). They were synthesized and characterized for sensing responses in solution. Cross-screening with the full set of metal ions showed that they have a wide variety of responses, including emission enhancements and red- and blue-shifts. The diversity of sensor responses allows as few as two sensors (1 and 2) to be used together to successfully differentiate these eight metals. As a test, a set of unknown metal ion solutions in blind studies were also successfully identified based on the response pattern of the sensors. The modular nature of the sensor design strategy suggests a broadly applicable approach to finding sensors for differentiating many different cations by pattern-based recognition, simply by varying the sequence and composition of ligands and fluorophores on a DNA synthesizer.

    View details for DOI 10.1021/ja109561e

    View details for Web of Science ID 000288291300053

    View details for PubMedID 21294558

  • Fluorescent xDNA nucleotides as efficient substrates for a template-independent polymerase NUCLEIC ACIDS RESEARCH Jarchow-Choy, S. K., Krueger, A. T., Liu, H., Gao, J., Kool, E. T. 2011; 39 (4): 1586-1594


    Template independent polymerases, and terminal deoxynucleotidyl transferase (TdT) in particular, have been widely used in enzymatic labeling of DNA 3'-ends, yielding fluorescently-labeled polymers. The majority of fluorescent nucleotides used as TdT substrates contain tethered fluorophores attached to a natural nucleotide, and can be hindered by undesired fluorescence characteristics such as self-quenching. We previously documented the inherent fluorescence of a set of four benzo-expanded deoxynucleoside analogs (xDNA) that maintain Watson-Crick base pairing and base stacking ability; however, their substrate abilities for standard template-dependent polymerases were hampered by their large size. However, it seemed possible that a template-independent enzyme, due to lowered geometric constraints, might be less restrictive of nucleobase size. Here, we report the synthesis and study of xDNA nucleoside triphosphates, and studies of their substrate abilities with TdT. We find that this polymerase can incorporate each of the four xDNA monomers with kinetic efficiencies that are nearly the same as those of natural nucleotides, as measured by steady-state methods. As many as 30 consecutive monomers could be incorporated. Fluorescence changes over time could be observed in solution during the enzymatic incorporation of expanded adenine (dxATP) and cytosine (dxCTP) analogs, and after incorporation, when attached to a glass solid support. For (dxA)(n) polymers, monomer emission quenching and long-wavelength excimer emission was observed. For (dxC)(n), fluorescence enhancement was observed in the polymer. TdT-mediated synthesis may be a useful approach for creating xDNA labels or tags on DNA, making use of the fluorescence and strong hybridization properties of the xDNA.

    View details for DOI 10.1093/nar/gkq853

    View details for Web of Science ID 000288019400043

    View details for PubMedID 20947563

  • Multispectral labeling of antibodies with polyfluorophores on a DNA backbone and application in cellular imaging PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Guo, J., Wang, S., Dai, N., Teo, Y. N., Kool, E. T. 2011; 108 (9): 3493-3498


    Most current approaches to multiantigen fluorescent imaging require overlaying of multiple images taken with separate filter sets as a result of differing dye excitation requirements. This requirement for false-color composite imaging prevents the user from visualizing multiple species in real time and disallows imaging of rapidly moving specimens. To address this limitation, here we investigate the use of oligodeoxyfluoroside (ODF) fluorophores as labels for antibodies. ODFs are short DNA-like oligomers with fluorophores replacing the DNA bases and can be assembled in many colors with excitation at a single wavelength. A DNA synthesizer was used to construct several short ODFs carrying a terminal alkyne group and having emission maxima of 410-670 nm. We developed a new approach to antibody conjugation, using Huisgen-Sharpless cycloaddition, which was used to react the alkynes on ODFs with azide groups added to secondary antibodies. Multiple ODF-tagged secondary antibodies were then used to mark primary antibodies. The set of antibodies was tested for spectral characteristics in labeling tubulin in HeLa cells and revealed a wide spectrum of colors, ranging from violet-blue to red with excitation through a single filter (340-380 nm). Selected sets of the differently labeled secondary antibodies were then used to simultaneously mark four antigens in fixed cells, using a single image and filter set. We also imaged different surface tumor markers on two live cell lines. Experiments showed that all colors could be visualized simultaneously by eye under the microscope, yielding multicolor images of multiple cellular antigens in real time.

    View details for DOI 10.1073/pnas.1017349108

    View details for Web of Science ID 000287844400014

    View details for PubMedID 21321224

  • The Components of xRNA: Synthesis and Fluorescence of a Full Genetic Set of Size-Expanded Ribonucleosides ORGANIC LETTERS Hernandez, A. R., Kool, E. T. 2011; 13 (4): 676-679


    The synthesis and properties of a full set of four benzo-expanded ribonucleosides (xRNA), analogous to A, G, C, and U RNA monomers, are described. The nucleosides are efficient fluorophores with emission maxima of 369-411 nm. The compounds are expected to be useful as RNA pathway probes and as components of an unnatural ribopolymer.

    View details for DOI 10.1021/ol102915f

    View details for Web of Science ID 000287122800034

    View details for PubMedID 21214222

  • Fluorescent DNA chemosensors: identification of bacterial species by their volatile metabolites CHEMICAL COMMUNICATIONS Koo, C., Wang, S., Gaur, R. L., Samain, F., Banaei, N., Kool, E. T. 2011; 47 (41): 11435-11437


    Polyfluorophores built on a DNA scaffold (ODFs) were synthesized and tested for fluorescence responses to the volatiles from M. tuberculosis, E. coli and P. putida in closed Petri dishes. Two sensors in a pattern-based response could distinguish the bacterial strains accurately, suggesting the use of ODFs in rapid identification of infectious agents.

    View details for DOI 10.1039/c1cc14871k

    View details for Web of Science ID 000295696300011

    View details for PubMedID 21935547

  • DNA polyfluorophores as highly diverse chemosensors of toxic gases CHEMICAL SCIENCE Koo, C., Samain, F., Dai, N., Kool, E. T. 2011; 2 (10): 1910-1917

    View details for DOI 10.1039/c1sc00301a

    View details for Web of Science ID 000294503900005

  • Protease Probes Built from DNA: Multispectral Fluorescent DNA-Peptide Conjugates as Caspase Chemosensors ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Dai, N., Guo, J., Teo, Y. N., Kool, E. T. 2011; 50 (22): 5105-5109

    View details for DOI 10.1002/anie.201007805

    View details for Web of Science ID 000290665100013

    View details for PubMedID 21455915

  • Fluorescent DNA-based enzyme sensors CHEMICAL SOCIETY REVIEWS Dai, N., Kool, E. T. 2011; 40 (12): 5756-5770


    Fluorescent sensors that make use of DNA structures have become widely useful in monitoring enzymatic activities. Early studies focused primarily on enzymes that naturally use DNA or RNA as the substrate. However, recent advances in molecular design have enabled the development of nucleic acid sensors for a wider range of functions, including enzymes that do not normally bind DNA or RNA. Nucleic acid sensors present some potential advantages over classical small-molecule sensors, including water solubility and ease of synthesis. An overview of the multiple strategies under recent development is presented in this critical review, and expected future developments in microarrays, single molecule analysis, and in vivo sensing are discussed (160 references).

    View details for DOI 10.1039/c0cs00162g

    View details for Web of Science ID 000296986000013

    View details for PubMedID 21290032

  • Differentiating a Diverse Range of Volatile Organic Compounds with Polyfluorophore Sensors Built on a DNA Scaffold CHEMISTRY-A EUROPEAN JOURNAL Samain, F., Dai, N., Kool, E. T. 2011; 17 (1): 174-183


    Oligodeoxyfluorosides (ODFs) are short DNA-like oligomers in which DNA bases are replaced with fluorophores. A preliminary study reported that some sequences of ODFs were able to respond to a few organic small molecules in the vapor phase, giving a change in fluorescence. Here, we follow up on this finding by investigating a larger range of volatile organic analytes, and a considerably larger set of sensors. A library of tetramer ODFs of 2401 different sequences was prepared by using combinatorial methods, and was screened in air for fluorescence responses to a set of ten different volatile organics, including multiple aromatic and aliphatic compounds, acids and bases, varied functional groups, and closely related structures. Nineteen responding sensors were selected and characterized. These sensors were cross-screened against all ten analytes, and responses were measured qualitatively (by changes in color and intensity) and quantitatively (by measuring ?R, ?G, and ?B values averaged over five to six sensor beads; R=red, G=green, B=blue). The results show that sensor responses were diverse, with a single sensor responding differently to as many as eight of the ten analytes; multiple classes of responses were seen, including quenching, lighting-up, and varied shifts in wavelength. Responses were strong, with raw ?R, ?G, and ?B values of as high as >200 on a 256-unit scale and unamplified changes in many cases apparent to the naked eye. Sensors were identified that could distinguish clearly between even very closely related compounds such as acrolein and acrylonitrile. Statistical methods were applied to select a small set of four sensors that, as a pattern response, could distinguish between all ten analytes with high confidence. Sequence analysis of the full set of sensors suggested that sequence/order of the monomer components, and not merely composition, was highly important in the responses.

    View details for DOI 10.1002/chem.201002836

    View details for Web of Science ID 000286682900019

    View details for PubMedID 21207614

  • Two Successive Reactions on a DNA Template: A Strategy for Improving Background Fluorescence and Specificity in Nucleic Acid Detection CHEMISTRY-A EUROPEAN JOURNAL Franzini, R. M., Kool, E. T. 2011; 17 (7): 2168-2175


    We report a new strategy for template-mediated fluorogenic chemistry that results in enhanced performance for the fluorescence detection of nucleic acids. In this approach, two successive templated reactions are required to induce a fluorescence signal, rather than only one. These novel fluorescein-labeled oligonucleotide probes, termed 2-STAR (STAR = Staudinger-triggered ?-azidoether release) probes, contain two quencher groups tethered by separate reductively cleavable linkers. When a 2-STAR quenched probe successively binds adjacent to two mono-triphenylphosphine-(TPP)-DNAs or one dual-TPP-DNA, the two quenchers are released, resulting in a fluorescence signal. Because of the requirement for two consecutive reactions, 2-STAR probes display an unprecedented level of sequence specificity for template-mediated probe designs. At the same time, background emission generated by off-template reactions or incomplete quenching is among the lowest of any fluorogenic reactive probes for the detection of DNA or RNA.

    View details for DOI 10.1002/chem.201002426

    View details for Web of Science ID 000287986500018

    View details for PubMedID 21294182

  • Two Successive Reactions on a DNA Template: A Strategy for Improving Background Fluorescence and Specificity in Nucleic Acid Detection. Chemistry (Weinheim an der Bergstrasse, Germany) Franzini, R. M., Kool, E. T. 2011


    We report a new strategy for template-mediated fluorogenic chemistry that results in enhanced performance for the fluorescence detection of nucleic acids. In this approach, two successive templated reactions are required to induce a fluorescence signal, rather than only one. These novel fluorescein-labeled oligonucleotide probes, termed 2-STAR (STAR=Staudinger-triggered ?-azidoether release) probes, contain two quencher groups tethered by separate reductively cleavable linkers. When a 2-STAR quenched probe successively binds adjacent to two mono-triphenylphosphine-(TPP)-DNAs or one dual-TPP-DNA, the two quenchers are released, resulting in a fluorescence signal. Because of the requirement for two consecutive reactions, 2-STAR probes display an unprecedented level of sequence specificity for template-mediated probe designs. At the same time, background emission generated by off-template reactions or incomplete quenching is among the lowest of any fluorogenic reactive probes for the detection of DNA or RNA.

    View details for PubMedID 21225606

  • Chemistry of nucleic acids: impacts in multiple fields CHEMICAL COMMUNICATIONS Khakshoor, O., Kool, E. T. 2011; 47 (25): 7018-7024


    Research in nucleic acids has made major advances in the past decade in multiple fields of science and technology. Here we discuss some of the most important findings in DNA and RNA research in the fields of biology, chemistry, biotechnology, synthetic biology, nanostructures and optical materials, with emphasis on how chemistry has impacted, and is impacted by, these developments. Major challenges ahead include the development of new chemical strategies that allow synthetically modified nucleic acids to enter into, and function in, living systems.

    View details for DOI 10.1039/c1cc11021g

    View details for Web of Science ID 000291613400001

    View details for PubMedID 21483917

  • Selective Sensor for Silver Ions Built From Polyfluorophores on a DNA Backbone ORGANIC LETTERS Tan, S. S., Teo, Y. N., Kool, E. T. 2010; 12 (21): 4820-4823


    To explore a new modular metal ion sensor design strategy, fluorophores and ligands were incorporated into short DNA-like oligomers. Compound 1 was found to function as a selective sensor for Ag(+) in aqueous buffer, where low micromolar concentrations of Ag(+) induce a red-shifted, turn-on fluorescence signal. Experiments with HeLa cells show that 1 can penetrate cells and yield a signal for intracellular Ag(+). This suggests a broadly applicable approach to developing sensors for a wide variety of cations.

    View details for DOI 10.1021/ol1019794

    View details for Web of Science ID 000283531000030

    View details for PubMedID 20883041

  • Templated Chemistry for Sequence-Specific Fluorogenic Detection of Duplex DNA CHEMBIOCHEM Li, H., Franzini, R. M., Bruner, C., Kool, E. T. 2010; 11 (15): 2132-2137


    We describe the development of templated fluorogenic chemistry for detection of specific sequences of duplex DNA in solution. In this approach, two modified homopyrimidine oligodeoxynucleotide probes are designed to bind by triple-helix formation at adjacent positions on a specific purine-rich target sequence of duplex DNA. One fluorescein-labeled probe contains an ?-azidoether linker to a fluorescence quencher; the second (trigger) probe carries a triarylphosphine group that is designed to reduce the azide and cleave the linker. The data showed that at pH 5.6 these probes yielded a strong fluorescence signal within minutes on addition to a complementary homopurine duplex DNA target. The signal increased by a factor of about 60, and was completely dependent on the presence of the target DNA. Replacement of cytosine in the probes with pseudoisocytosine allowed the templated chemistry to proceed readily at pH 7. Single nucleotide mismatches in the target oligonucleotide slowed the templated reaction considerably; this demonstrated high sequence selectivity. The use of templated fluorogenic chemistry for detection of duplex DNAs has not been previously reported and could allow detection of double-stranded DNA, at least for homopurine-homopyrimidine target sites, under native and nondenaturing conditions.

    View details for DOI 10.1002/cbic.201000329

    View details for Web of Science ID 000284050000013

    View details for PubMedID 20859985

  • Probing the Interaction of Archaeal DNA Polymerases with Deaminated Bases Using X-ray Crystallography and Non-Hydrogen Bonding Isosteric Base Analogues BIOCHEMISTRY Killelea, T., Ghosh, S., Tan, S. S., Heslop, P., Firbank, S. J., Kool, E. T., Connolly, B. A. 2010; 49 (27): 5772-5781


    Archaeal family-B DNA polymerases stall replication on encountering the pro-mutagenic bases uracil and hypoxanthine. This publication describes an X-ray crystal structure of Thermococcus gorgonarius polymerase in complex with a DNA containing hypoxanthine in the single-stranded region of the template, two bases ahead of the primer-template junction. Full details of the specific recognition of hypoxanthine are revealed, allowing a comparison with published data that describe uracil binding. The two bases are recognized by the same pocket, in the N-terminal domain, and make very similar protein-DNA interactions. Specificity for hypoxanthine (and uracil) arises from a combination of polymerase-base hydrogen bonds and shape fit between the deaminated bases and the pocket. The structure with hypoxanthine at position 2 explains the stimulation of the polymerase 3'-5' proofreading exonuclease, observed with deaminated bases at this location. A beta-hairpin element, involved in partitioning the primer strand between the polymerase and exonuclease active sites, inserts between the two template bases at the extreme end of the double-stranded DNA. This denatures the two complementary primer bases and directs the resulting 3' single-stranded extension toward the exonuclease active site. Finally, the relative importance of hydrogen bonding and shape fit in determining selectivity for deaminated bases has been examined using nonpolar isosteres. Affinity for both 2,4-difluorobenzene and fluorobenzimidazole, non-hydrogen bonding shape mimics of uracil and hypoxanthine, respectively, is strongly diminished, suggesting polar protein-base contacts are important. However, residual interaction with 2,4-difluorobenzene is seen, confirming a role for shape recognition.

    View details for DOI 10.1021/bi100421r

    View details for Web of Science ID 000279389300017

    View details for PubMedID 20527806

  • Toward a designed genetic system with biochemical function: polymerase synthesis of single and multiple size-expanded DNA base pairs ORGANIC & BIOMOLECULAR CHEMISTRY Lu, H., Krueger, A. T., Gao, J., Liu, H., Kool, E. T. 2010; 8 (12): 2704-2710


    The development of alternative architectures for genetic information-encoding systems offers the possibility of new biotechnological tools as well as basic insights into the function of the natural system. In order to examine the potential of benzo-expanded DNA (xDNA) to encode and transfer biochemical information, we carried out a study of the processing of single xDNA pairs by DNA Polymerase I Klenow fragment (Kf, an A-family sterically rigid enzyme) and by the Sulfolobus solfataricus polymerase Dpo4 (a flexible Y-family polymerase). Steady-state kinetics were measured and compared for enzymatic synthesis of the four correct xDNA pairs and twelve mismatched pairs, by incorporation of dNTPs opposite single xDNA bases. Results showed that, like Kf, Dpo4 in most cases selected the correctly paired partner for each xDNA base, but with efficiency lowered by the enlarged pair size. We also evaluated kinetics for extension by these polymerases beyond xDNA pairs and mismatches, and for exonuclease editing by the Klenow exo+ polymerase. Interestingly, the two enzymes were markedly different: Dpo4 extended pairs with relatively high efficiencies (within 18-200-fold of natural DNA), whereas Kf essentially failed at extension. The favorable extension by Dpo4 was tested further by stepwise synthesis of up to four successive xDNA pairs on an xDNA template.

    View details for DOI 10.1039/c002766a

    View details for Web of Science ID 000278964600006

    View details for PubMedID 20407680

  • Double Displacement: An Improved Bioorthogonal Reaction Strategy for Templated Nucleic Acid Detection BIOCONJUGATE CHEMISTRY Kleinbaum, D. J., Miller, G. P., Kool, E. T. 2010; 21 (6): 1115-1120


    Quenched autoligation probes have been employed previously in a target-templated nonenzymatic ligation strategy for detecting nucleic acids in cells by fluorescence. A common source of background signal in such probes is the undesired reaction with water and other cellular nucleophiles. Here, we describe a new class of self-ligating probes, double displacement (DD) probes, that rely on two displacement reactions to fully unquench a nearby fluorophore. Three potential double displacement architectures, all possessing two fluorescence quencher/leaving groups (dabsylate groups), were synthesized and evaluated for templated reaction with nucleophile (phosphorothioate) probes both in vitro and in intact bacterial cells. All three DD probe designs provided substantially better initial quenching than a single-Dabsyl control. In isothermal templated reactions in vitro, double displacement probes yielded considerably lower background signal than previous single displacement probes; investigation into the mechanism revealed that one dabsylate acts as a sacrificial leaving group, reacting nonspecifically with water, but yielding little signal because another quencher group remains. Templated reaction with the specific nucleophile probe is required to activate a signal. The double displacement probes provided a ca. 80-fold turn-on signal and yielded a 2-4-fold improvement in signal/background over single Dabsyl probes. The best-performing probe architecture was demonstrated in a two-color, FRET-based two-allele discrimination system in vitro and was shown to be capable of discriminating between two closely related species of bacteria differing by a single nucleotide at an rRNA target site.

    View details for DOI 10.1021/bc100165h

    View details for Web of Science ID 000278734900016

    View details for PubMedID 20509625

  • Polyfluorophores on a DNA Backbone: Sensors of Small Molecules in the Vapor Phase ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Samain, F., Ghosh, S., Teo, Y. N., Kool, E. T. 2010; 49 (39): 7025-7029

    View details for DOI 10.1002/anie.201002701

    View details for Web of Science ID 000282477800016

    View details for PubMedID 20721995

  • DNA-polyfluorophore excimers as sensitive reporters for esterases and lipases CHEMICAL COMMUNICATIONS Dai, N., Teo, Y. N., Kool, E. T. 2010; 46 (8): 1221-1223


    DNA-scaffolded oligodeoxyriboside fluorophores (ODFs) were used as the reporters in turn-on sensing of enzymatic bond-cleaving activity. A tetramer ODF of pyrene deoxynucleosides displayed high quenching efficiency when conjugated via ester linkages with a dabcyl quencher, and yielded large signal increases with several enzymes in vitro and in intact human cells.

    View details for DOI 10.1039/b926338a

    View details for Web of Science ID 000274396700008

    View details for PubMedID 20449256

  • Differentiating a Diverse Range of Volatile Organic Compounds with Polyfluorophore Sensors Built on a DNA Scaffold. Chemistry (Weinheim an der Bergstrasse, Germany) Samain, F., Dai, N., Kool, E. T. 2010


    Oligodeoxyfluorosides (ODFs) are short DNA-like oligomers in which DNA bases are replaced with fluorophores. A preliminary study reported that some sequences of ODFs were able to respond to a few organic small molecules in the vapor phase, giving a change in fluorescence. Here, we follow up on this finding by investigating a larger range of volatile organic analytes, and a considerably larger set of sensors. A library of tetramer ODFs of 2401 different sequences was prepared by using combinatorial methods, and was screened in air for fluorescence responses to a set of ten different volatile organics, including multiple aromatic and aliphatic compounds, acids and bases, varied functional groups, and closely related structures. Nineteen responding sensors were selected and characterized. These sensors were cross-screened against all ten analytes, and responses were measured qualitatively (by changes in color and intensity) and quantitatively (by measuring ?R, ?G, and ?B values averaged over five to six sensor beads; R=red, G=green, B=blue). The results show that sensor responses were diverse, with a single sensor responding differently to as many as eight of the ten analytes; multiple classes of responses were seen, including quenching, lighting-up, and varied shifts in wavelength. Responses were strong, with raw ?R, ?G, and ?B values of as high as >200 on a 256-unit scale and unamplified changes in many cases apparent to the naked eye. Sensors were identified that could distinguish clearly between even very closely related compounds such as acrolein and acrylonitrile. Statistical methods were applied to select a small set of four sensors that, as a pattern response, could distinguish between all ten analytes with high confidence. Sequence analysis of the full set of sensors suggested that sequence/order of the monomer components, and not merely composition, was highly important in the responses.

    View details for PubMedID 21154704

  • Sandwich probes: two simultaneous reactions for templated nucleic acid detection CHEMICAL COMMUNICATIONS Kleinbaum, D. J., Kool, E. T. 2010; 46 (43): 8154-8156


    Fluorescence-quenched nucleic acid probes with reactive moieties at both the 5' and 3' ends are synthesized and tested for reaction with two adjacent nucleophile-containing DNAs. These probes improve signal to background over singly reactive probes and can discriminate single nucleotide polymorphisms in the target DNA or RNA.

    View details for DOI 10.1039/c0cc01968b

    View details for Web of Science ID 000283495500007

    View details for PubMedID 20927470

  • Polyfluorophore Excimers and Exciplexes as FRET Donors in DNA BIOCONJUGATE CHEMISTRY Teo, Y. N., Kool, E. T. 2009; 20 (12): 2371-2380


    We describe studies aimed at testing whether oligomeric exciplex and excimer fluorophores conjugated to DNA have the potential to act as donors for energy transfer by the Forster mechanism. Oligodeoxyfluorosides (ODFs) are composed of stacked, electronically interacting fluorophores replacing the bases on a DNA scaffold. The monomer chromophores in the twenty tetramer-length ODFs studied here include pyrene (Y), benzopyrene (B), perylene (E), dimethylaminostilbene (D), and a nonfluorescent spacer (S); these are conjugated in varied combinations at the 3' end of a 14mer DNA probe sequence. In the absence of an acceptor chromophore, many of the ODF-DNAs show broad, unstructured long-wavelength emission peaks characteristic of excimer and exciplex excited states, similar to what has been observed for unconjugated ODFs. Although such delocalized excited states have been widely studied, we know of no prior report of their use in FRET. We tested the ability of the twenty ODFs to donate energy to Cy5 and TAMRA dyes conjugated to a complementary strand of DNA, with these acceptors oriented either at the near or far end of the ODF-conjugated probes. Results showed that a number of the ODF fluorophores exhibited relatively efficient energy transfer characteristic of the Forster mechanism, as judged by drops in donor emission quantum yield and fluorescence lifetime, accompanied by increases in intensity of acceptor emission bands. Excimer/exciplex bands in the donors were selectively quenched while shorter-wavelength monomer emission stayed relatively constant, consistent with the notion that the delocalized excited states, rather than individual fluorophores, are the donors. Interestingly, only specific sequences of ODFs were able to act as donors, while others did not, even though their emission wavelengths were similar. The new FRET donors possess large Stokes shifts, which can be beneficial for multiple applications. In addition, all ODFs can be excited at a single wavelength; thus, ODFs may be candidates as "universal FRET donors", thus allowing multicolor FRET of multiple species to be carried out with one excitation.

    View details for DOI 10.1021/bc9003926

    View details for Web of Science ID 000272690100022

    View details for PubMedID 19916519

  • Efficient Nucleic Acid Detection by Templated Reductive Quencher Release JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Franzini, R. M., Kool, E. T. 2009; 131 (44): 16021-?


    RNA-templated fluorescence activation is a nucleic acid detection strategy that offers the possibility of direct visual detection of genetic information in living cells. Here we describe a new reaction strategy for fluorescence activation in which a phosphine on one DNA probe reduces an azide group in a linker on a second probe, resulting in linker cleavage and release of a fluorescence quenching group. These "Q-STAR" probes are shown to yield a strong fluorescence turn-on signal in approximately 20 min, with very low background and substantial amplification by turnover on the template. A green/red pair of such probes allowed the discrimination of two bacterial species by a single nucleotide difference in their 16S rRNA. The beneficial properties of the reductive quencher release design make these probes promising candidates for widespread application in the detection of nucleic acids in vitro and in cells.

    View details for DOI 10.1021/ja904138v

    View details for Web of Science ID 000271513700028

    View details for PubMedID 19886694

  • Evolving a Polymerase for Hydrophobic Base Analogues JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Loakes, D., Gallego, J., Pinheiro, V. B., Kool, E. T., Holliger, P. 2009; 131 (41): 14827-14837


    Hydrophobic base analogues (HBAs) have shown great promise for the expansion of the chemical and coding potential of nucleic acids but are generally poor polymerase substrates. While extensive synthetic efforts have yielded examples of HBAs with favorable substrate properties, their discovery has remained challenging. Here we describe a complementary strategy for improving HBA substrate properties by directed evolution of a dedicated polymerase using compartmentalized self-replication (CSR) with the archetypal HBA 5-nitroindole (d5NI) and its derivative 5-nitroindole-3-carboxamide (d5NIC) as selection substrates. Starting from a repertoire of chimeric polymerases generated by molecular breeding of DNA polymerase genes from the genus Thermus, we isolated a polymerase (5D4) with a generically enhanced ability to utilize HBAs. The selected polymerase. 5D4 was able to form and extend d5NI and d5NIC (d5NI(C)) self-pairs as well as d5NI(C) heteropairs with all four bases with efficiencies approaching, or exceeding, those of the cognate Watson-Crick pairs, despite significant distortions caused by the intercalation of the d5NI(C) heterocycles into the opposing strand base stack, as shown by nuclear magnetic resonance spectroscopy (NMR). Unlike Taq polymerase, 5D4 was also able to extend HBA pairs such as Pyrene: varphi (abasic site), d5NI: varphi, and isocarbostyril (ICS): 7-azaindole (7AI), allowed bypass of a chemically diverse spectrum of HBAs, and enabled PCR amplification with primers comprising multiple d5NI(C)-substitutions, while maintaining high levels of catalytic activity and fidelity. The selected polymerase 5D4 promises to expand the range of nucleobase analogues amenable to replication and should find numerous applications, including the synthesis and replication of nucleic acid polymers with expanded chemical and functional diversity.

    View details for DOI 10.1021/ja9039696

    View details for Web of Science ID 000271271800055

    View details for PubMedID 19778048

  • Structure and Replication of yDNA: A Novel Genetic Set Widened by Benzo-Homologation CHEMBIOCHEM Lu, H., Lynch, S. R., Lee, A. H., Kool, E. T. 2009; 10 (15): 2530-2538


    In a functioning genetic system, the information-encoding molecule must form a regular self-complementary complex (for example, the base-paired double helix of DNA) and it must be able to encode information and pass it on to new generations. Here we study a benzo-widened DNA-like molecule (yDNA) as a candidate for an alternative genetic set, and we explicitly test these two structural and functional requirements. The solution structure of a 10 bp yDNA duplex is measured by using 2D-NMR methods for a simple sequence composed of T-yA/yA-T pairs. The data confirm an antiparallel, right-handed, hydrogen-bonded helix resembling B-DNA but with a wider diameter and enlarged base-pair size. In addition to this, the abilities of two different polymerase enzymes (Klenow fragment of DNA pol I (Kf) and the repair enzyme Dpo4) to synthesize and extend the yDNA pairs T-yA, A-yT, and G-yC are measured by steady-state kinetics studies. Not surprisingly, insertion of complementary bases opposite yDNA bases is inefficient due to the larger base-pair size. We find that correct pairing occurs in several cases by both enzymes, but that common and relatively efficient mispairing involving T-yT and T-yC pairs interferes with fully correct formation and extension of pairs by these polymerases. Interestingly, the data show that extension of the large pairs is considerably more efficient with the flexible repair enzyme (Dpo4) than with the more rigid Kf enzyme. The results shed light on the properties of yDNA as a candidate for an alternative genetic information-encoding molecule and as a tool for application in basic science and biomedicine.

    View details for DOI 10.1002/cbic.200900434

    View details for Web of Science ID 000271095600015

    View details for PubMedID 19780073

  • Nonpolar Nucleoside Mimics as Active Substrates for Human Thymidine Kinases JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Jarchow-Choy, S. K., Sjuvarsson, E., Sintim, H. O., Eriksson, S., Kool, E. T. 2009; 131 (15): 5488-5494


    We describe the use of nonpolar nucleoside analogues of systematically varied size and shape to probe the mechanisms by which the two human thymidine kinases (TK1 and TK2) recognize and phosphorylate their substrate, thymidine. Comparison of polar thymidine with a nonpolar isostere, 2,4-difluorotoluene deoxyriboside, as substrates for the two enzymes establishes that TK1 requires electrostatic complementarity to recognize the thymine base with high efficiency. Conversely, TK2 does not and phosphorylates the hydrophobic shape mimic with efficiency nearly the same as the natural substrate. To test the response to nucleobase size, thymidine-like analogues were systematically varied by replacing the 2,4 substituents on toluene with hydrogen and the halogen series (H, F, Cl, Br, I). Both enzymes showed a distinct preference for substrates having the natural size. To examine the shape preference, we prepared four mono- and difluorotoluene deoxyribosides with varying positions of substitutions. While TK1 did not accept these nonpolar analogues as substrates, TK2 did show varying levels of phosphorylation of the shape-varied set. This latter enzyme preferred toluene nucleoside analogues having steric projections at the 2 and 4 positions, as is found in thymine, and strongly disfavored substitution at the 3-position. Steady-state kinetics measurements showed that the 4-fluoro compound (7) had an apparent V(max)/K(m) value within 14-fold of the natural substrate, and the 2,4-difluoro compound (1), which is the closest isostere of thymidine, had a value within 2.5-fold. The results establish that nucleoside recognition mechanisms for the two classes of enzymes are very different. On the basis of these data, nonpolar nucleosides are likely to be active in the nucleotide salvage pathway in human cells, suggesting new designs for future bioactive molecules.

    View details for DOI 10.1021/ja808244t

    View details for Web of Science ID 000265268100031

    View details for PubMedID 20560637

  • Redesigning the Architecture of the Base Pair: Toward Biochemical and Biological Function of New Genetic Sets CHEMISTRY & BIOLOGY Krueger, A. T., Kool, E. T. 2009; 16 (3): 242-248


    Recognition of the nucleic acid bases within the DNA scaffold comprises the basis for transmission of genetic information, dictating protein and cell assembly, organismal development, and evolution. Driven in part by the need to test our current understanding of this information transfer, chemists have begun to design and synthesize nonnatural bases and base pair structures to mimic the function of DNA without relying on Nature's purine-pyrimidine base pair scaffold. Multiple examples have been recently described that self-assemble stably and sequence specifically in vitro, and some isolated unnatural base pairs can be replicated in vitro as well. Moreover, recent experiments with unnatural bases in bacterial cells have demonstrated surprisingly efficient reading of the chemical information. This suggests the future possibility of redesigning and replacing the chemical information of an evolving cell while retaining biological function.

    View details for DOI 10.1016/j.chembiol.2008.12.004

    View details for Web of Science ID 000264859200003

    View details for PubMedID 19318205

  • Polyfluorophores on a DNA Backbone: A Multicolor Set of Labels Excited at One Wavelength JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Teo, Y. N., Wilson, J. N., Kool, E. T. 2009; 131 (11): 3923-3933


    We recently described the assembly of fluorescent deoxyriboside monomers ("fluorosides") into DNA-like phosphodiester oligomers (oligodeoxyfluorosides or ODFs) in which hydrocarbon and heterocyclic aromatic fluorophores interact both physically and electronically. Here we report the identification of a multicolor set of water-soluble ODF dyes that display emission colors across the visible spectrum, and all of which can be simultaneously excited by long-wavelength UV light at 340-380 nm. Multispectral dye candidates were chosen from a library of 4096 tetramer ODFs constructed on PEG-polystyrene beads using a simple long-pass filter to observe all visible colors at the same time. We resynthesized and characterized a set of 23 ODFs containing one to four individual chromophores and included 2-3 spacer monomers to increase aqueous solubility and minimize aggregation. Emission maxima of this set range from 376 to 633 nm, yielding apparent colors from violet to red, all of which can be visualized directly. The spectra of virtually all ODFs in this set varied considerably from the simple combination of monomer components, revealing extensive electronic interactions between the presumably stacked monomers. In addition, comparisons of anagrams in the set (isomers having the same components in a different sequence) reveal the importance of nearest-neighbor interactions in the emissive behavior. Preliminary experiments with human tumor (HeLa) cells, observing two ODFs by laser confocal microscopy, showed that they can penetrate the outer cellular membrane, yielding cytoplasmic localization. In addition, a set of four distinctly colored ODFs was incubated with live zebrafish embryos, showing tissue penetration, apparent biostability, and no apparent toxicity. The results suggest that ODF dyes may be broadly useful as labels in biological systems, allowing the simultaneous tracking of multiple species by color, and allowing visualization in moving systems where classical fluorophores fail.

    View details for DOI 10.1021/ja805502k

    View details for Web of Science ID 000264792700048

    View details for PubMedID 19254023

  • RNA major groove modifications improve siRNA stability and biological activity NUCLEIC ACIDS RESEARCH Terrazas, M., Kool, E. T. 2009; 37 (2): 346-353


    RNA 5-methyl and 5-propynyl pyrimidine analogs were substituted into short interfering RNAs (siRNAs) to probe major groove steric effects in the active RNA-induced silencing complex (RISC). Synthetic RNA guide strands containing varied combinations of propynyl and methyl substitution revealed that all C-5 substitutions increased the thermal stability of siRNA duplexes containing them. Cellular gene suppression experiments using luciferase targets in HeLa cells showed that the bulky 5-propynyl modification was detrimental to RNA interference activity, despite its stabilization of the helix. Detrimental effects of this substitution were greatest at the 5'-half of the guide strand, suggesting close steric approach of proteins in the RISC complex with that end of the siRNA/mRNA duplex. However, substitutions with the smaller 5-methyl group resulted in gene silencing activities comparable to or better than that of wild-type siRNA. The major groove modifications also increased the serum stability of siRNAs.

    View details for DOI 10.1093/nar/gkn958

    View details for Web of Science ID 000262963400012

    View details for PubMedID 19042976

  • Polyfluorophore Labels on DNA: Dramatic Sequence Dependence of Quenching CHEMISTRY-A EUROPEAN JOURNAL Teo, Y. N., Wilson, J. N., Kool, E. T. 2009; 15 (43): 11551-11558


    We describe studies carried out in the DNA context to test how a common fluorescence quencher, dabcyl, interacts with oligodeoxynucleoside fluorophores (ODFs)--a system of stacked, electronically interacting fluorophores built on a DNA scaffold. We tested twenty different tetrameric ODF sequences containing varied combinations and orderings of pyrene (Y), benzopyrene (B), perylene (E), dimethylaminostilbene (D), and spacer (S) monomers conjugated to the 3' end of a DNA oligomer. Hybridization of this probe sequence to a dabcyl-labeled complementary strand resulted in strong quenching of fluorescence in 85% of the twenty ODF sequences. The high efficiency of quenching was also established by their large Stern-Volmer constants (K(SV)) of between 2.1 x 10(4) and 4.3 x 10(5) M(-1), measured with a free dabcyl quencher. Interestingly, quenching of ODFs displayed strong sequence dependence. This was particularly evident in anagrams of ODF sequences; for example, the sequence BYDS had a K(SV) that was approximately two orders of magnitude greater than that of BSDY, which has the same dye composition. Other anagrams, for example EDSY and ESYD, also displayed different responses upon quenching by dabcyl. Analysis of spectra showed that apparent excimer and exciplex emission bands were quenched with much greater efficiency compared to monomer emission bands by at least an order of magnitude. This suggests an important role played by delocalized excited states of the pi stack of fluorophores in the amplified quenching of fluorescence.

    View details for DOI 10.1002/chem.200901607

    View details for Web of Science ID 000271992100018

    View details for PubMedID 19780115

  • Efficient Replication Bypass of Size-Expanded DNA Base Pairs in Bacterial Cells ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Delaney, J. C., Gao, J., Liu, H., Shrivastav, N., Essigmann, J. M., Kool, E. T. 2009; 48 (25): 4524-4527


    Supersize me! Size-expanded DNA bases (xDNA) are able to encode natural DNA sequences in replication. In vitro experiments with a DNA polymerase show nucleotide incorporation opposite the xDNA bases with correct pairing. In vivo experiments using E. coli show that two xDNA bases (xA and xC, see picture) encode the correct replication partners.

    View details for DOI 10.1002/anie.200805683

    View details for Web of Science ID 000267305300014

    View details for PubMedID 19444841

  • Polymerase Amplification, Cloning, and Gene Expression of Benzo-Homologous "yDNA" Base Pairs CHEMBIOCHEM Chelliserrykattil, J., Lu, H., Lee, A. H., Kool, E. T. 2008; 9 (18): 2976-2980


    A widened DNA base-pair architecture is studied in an effort to explore the possibility of whether new genetic system designs might possess some of the functions of natural DNA. In the "yDNA" system, pairs are homologated by addition of a benzene ring, which yields (in the present study) benzopyrimidines that are correctly paired with purines. Here we report initial tests of ability of the benzopyrimidines yT and yC to store and transfer biochemical and biological information in vitro and in bacterial cells. In vitro primer extension studies with two polymerases showed that the enzymes could insert the correct nucleotides opposite these yDNA bases, but with low selectivity. PCR amplifications with a thermostable polymerase resulted in correct pairings in 15-20 % of the cases, and more successfully when yT or yC were situated within the primers. Segments of DNA containing one or two yDNA bases were then ligated into a plasmid and tested for their ability to successfully lead the expression of an active protein in vivo. Although active at only a fraction of the activity of fully natural DNA, the unnatural bases encoded the correct codon bases in the majority of cases when singly substituted, and yielded functioning green fluorescent protein. Although the activities with native polymerases are modest with these large base pairs, this is the first example of encoding protein in vivo by an unnatural DNA base pair architecture.

    View details for DOI 10.1002/cbic.200800339

    View details for Web of Science ID 000261876900013

    View details for PubMedID 19053129

  • 7-Azidomethoxy-Coumarins as Profluorophores for Templated Nucleic Acid Detection CHEMBIOCHEM Franzini, R. M., Kool, E. T. 2008; 9 (18): 2981-2988


    Templated nucleic acid detection is an emerging bioanalytical method that makes use of the target DNA or RNA strand to initiate a fluorogenic reaction. The Staudinger reduction holds particular promise for templated sensing of nucleic acids because the involved functional groups are highly chemoselective. Here, the azidomethoxy group, which can be removed under Staudinger conditions, is used to cage 7-hydroxycoumarin fluorophores. Reduction by phosphines and subsequent loss of the azidomethoxy substituent induce a significant bathochromic shift of the major absorbance band in the near UV region. When excited at the appropriate wavelength, this change in the absorbance spectrum translates into a substantial fluorescence turn-on signal. The described profluorophores are readily conjugated to amino-modified DNAs and are rapidly uncaged by a triphenylphosphine-DNA probe under the control of a DNA template. In addition, turnover of the probes on the target strand occurs and yields substantial signal amplification.

    View details for DOI 10.1002/cbic.200800507

    View details for Web of Science ID 000261876900014

    View details for PubMedID 19035374

  • Visualization of Long Human Telomere Mimics by Single-Molecule Fluorescence Imaging JOURNAL OF PHYSICAL CHEMISTRY B Pomerantz, A. K., Moerner, W. E., Kool, E. T. 2008; 112 (42): 13184-13187


    Study of long single-stranded telomeric DNA is important for a variety of basic science and biotechnological applications, yet few methods exist for synthesis and visualization of single copies of this DNA in solution at biologically relevant length scales necessary for assessment of heterogeneity in its structure and behavior. We have synthesized kilobase-long single-stranded human telomere mimics in situ by rolling circle replication (RCR) on a microscope coverslip surface and visualized individual strands by staining with SYBR Gold. Under buffer flow, differential extensibility and varying morphology of these long telomere-mimicking DNA sequences were observed at the single-molecule level in real time. Using this procedure, we detected striking differences in the extensibility of individual RCR products based on the human G-rich telomeric sequence in the presence and absence of short, complementary single-stranded oligonucleotides. We also apply this new mode of single-stranded DNA characterization to probe the interaction of kilobase-length telomere mimics with the small-molecule G-quadruplex-binding agent TMPyP4.

    View details for DOI 10.1021/jp806696u

    View details for Web of Science ID 000260100900005

    View details for PubMedID 18817431

  • Organometallic activation of a fluorogen for templated nucleic acid detection ORGANIC LETTERS Franzini, R. M., Kool, E. T. 2008; 10 (14): 2935-2938


    A nucleic acid detection scheme that employs DNA-mediated delivery of an organomercury activator to unmask a fluorophore is described. The approach relies on adjacent hybridization of two oligonucleotide conjugates containing organomercury and caged rhodamine functionalities. Postsynthetic conjugation of amino-modified DNAs enabled efficient preparation of these probes. Complementary DNA templates yielded fluorescence signals arising from metal-assisted rhodamine uncaging.

    View details for DOI 10.1021/ol800878b

    View details for Web of Science ID 000257629200005

    View details for PubMedID 18549220

  • Importance of hydrogen bonding for efficiency and specificity of the human mitochondrial DNA polymerase JOURNAL OF BIOLOGICAL CHEMISTRY Lee, H. R., Helquist, S. A., Kool, E. T., Johnson, K. A. 2008; 283 (21): 14402-14410


    To assess the contribution to discrimination afforded by base pair hydrogen bonding during DNA replication by the human mitochondrial DNA polymerase, we examined nucleoside mimics lacking hydrogen bond forming capability but retaining the overall steric shape of the natural nucleotide. We employed oligonucleotide templates containing either a deoxyadenosine shape mimic (dQ) or a deoxythymidine shape mimic (dF). Additionally, the nucleoside triphosphate analogs difluorotoluene deoxynucleoside triphosphate, 9-methyl-1-H-imidazo[(4,5)-b]pyridine deoxyribose triphosphate, and 4-methylbenzimidazole deoxyribose triphosphate (dZTP; another dATP shape mimic) were assayed. We used pre-steady state methods to determine the kinetic parameters governing nucleotide incorporation, k(pol) and K(d). In general, the loss of hydrogen bonding potential led to 2-3 kcal/mol reduction in ground state binding free energy, whereas effects on the maximum rate of polymerization were quite variable, ranging from negligible (dATP:dF) to nearly 4 kcal/mol (dZTP:dT). Although we observed only a 46-fold reduction in discrimination when dF was present in the template, there was a complete elimination of discrimination when dQ was present in the template. Our data with dF indicate that hydrogen bonding contributes 2.2 kcal/mol toward the efficiency of incorporation, whereas data with dQ (which may overestimate the effect due to poor steric mimicry) suggest a contribution of up to 6.8 kcal/mol. Taken together, the data suggest that sterics are necessary but not sufficient to achieve optimal efficiency and fidelity for DNA polymerase. Base pair hydrogen bonding contributes at least a third of the energy underlying nucleoside incorporation efficiency and specificity.

    View details for DOI 10.1074/jbc.M705007200

    View details for Web of Science ID 000255941400027

    View details for PubMedID 17650502

  • Base pair hydrogen bonds are essential for proofreading selectivity by the human mitochondrial DNA polymerase JOURNAL OF BIOLOGICAL CHEMISTRY Lee, H. R., Helquist, S. A., Kool, E. T., Johnson, K. A. 2008; 283 (21): 14411-14416


    We have characterized the role of Watson-Crick hydrogen bonding in the 3'-terminal base pair on the 3'-5' exonuclease activity of the human mitochondrial DNA polymerase. Nonpolar nucleoside analogs of thymidine (dF) and deoxyadenosine (dQ) were used to eliminate hydrogen bonds while maintaining base pair size and shape. Exonuclease reactions were examined using pre-steady state kinetic methods. The time dependence of removal of natural nucleotides from the primer terminus paired opposite the nonpolar analogs dF and dQ were best fit to a double exponential function. The double exponential kinetics as well as the rates of excision (3-6 s(-1) fast phase, 0.16-0.3 s(-1) slow phase) are comparable with those observed during mismatch removal of natural nucleotides even when the analog was involved in a sterically correct base pair. Additionally, incorporation of the next correct base beyond a nonpolar analog was slow (0.04-0.22 s(-1)), so that more than 95% of terminal base pairs were removed rather than extended. The polymerase responds to all 3'-terminal base pairs containing a nonpolar analog as if it were a mismatch regardless of the identity of the paired base, and kinetic partitioning between polymerase and exonuclease sites failed to discriminate between correct and incorrect base pairs. Thus, sterics alone are insufficient, whereas hydrogen bond formation is essential for proper proofreading selectivity by the mitochondrial polymerase. The enzyme may use the alignment and prevention of fraying provided by proper hydrogen bonding and minor groove hydrogen bonding interactions as critical criteria for correct base pair recognition.

    View details for DOI 10.1074/jbc.M705006200

    View details for Web of Science ID 000255941400028

    View details for PubMedID 17650503

  • Probing the active site steric flexibility of HIV-1 reverse transcriptase: Different constraints for DNA- versus RNA-templated synthesist BIOCHEMISTRY Silverman, A. P., Garforth, S. J., Prasad, V. R., Kool, E. T. 2008; 47 (16): 4800-4807


    The steric flexibility or rigidity of polymerase active sites may play an important role in their fidelity of nucleic acid synthesis. In this regard, reverse transcriptases offer an unusual opportunity to compare two enzymatic activities that proceed in the same active site. For HIV-1 reverse transcriptase, reverse transcription (RNA-templated synthesis) is known to proceed with lower fidelity than DNA-templated synthesis. Here, we describe the use of a set of variably sized nonpolar thymidine and uracil mimics as molecular rulers to probe the active site steric constraints of HIV-1 RT, and for the first time, we directly compare the functional flexibility of these two activities. Steady-state kinetics of incorporation for natural dNTPs opposite unnatural template bases as well as for unnatural dNTPs opposite natural template bases are reported for the DNA-templated DNA synthesis, and comparison is made with recent data for the RNA-templated activity. Kinetics for extension beyond a base pair containing the analogue template bases are also reported both for RNA and DNA templates. Our results show that the DNA-dependent polymerization by HIV-RT is highly sensitive to size, strongly biasing against both too-small and too-large base pairs, while, by contrast, the RNA-dependent polymerization is only biased against analogues that are too small, and is much more accepting of larger base pairs. In addition, base pair extension with HIV-RT is found to be relatively insensitive to varied base pair size, consistent with its high mutagenicity. Overall, the data show greater rigidity with a DNA template as compared with an RNA template, which correlates directly with the higher fidelity of the DNA-templated synthesis. Possible structural explanations for these differences are discussed. We also report kinetics data for two HIV-1 RT mutants reported to have altered fidelity (F61A and K65R) using DNA templates containing nonpolar base analogues, and find that one of these (F61A) is a high-fidelity enzyme that appears to be sensitive to a loss of hydrogen-bonding groups.

    View details for DOI 10.1021/bi702427y

    View details for Web of Science ID 000254907800026

    View details for PubMedID 18366188

  • Fluorescence of size-expanded DNA bases: Reporting on DNA sequence and structure with an unnatural genetic set JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Krueger, A. T., Kool, E. T. 2008; 130 (12): 3989-3999


    We recently described the synthesis and helix assembly properties of expanded DNA (xDNA), which contains base pairs 2.4 A larger than natural DNA pairs. This designed genetic set is under study with the goals of mimicking the functions of the natural DNA-based genetic system and of developing useful research tools. Here, we study the fluorescence properties of the four expanded bases of xDNA (xA, xC, xG, xT) and evaluate how their emission varies with changes in oligomer length, composition, and hybridization. Experiments were carried out with short oligomers of xDNA nucleosides conjugated to a DNA oligonucleotide, and we investigated the effects of hybridizing these fluorescent oligomers to short complementary DNAs with varied bases opposite the xDNA bases. As monomer nucleosides, the xDNA bases absorb light in two bands: one at approximately 260 nm (similar to DNA) and one at longer wavelength ( approximately 330 nm). All are efficient violet-blue fluorophores with emission maxima at approximately 380-410 nm and quantum yields (Phifl) of 0.30-0.52. Short homo-oligomers of the xDNA bases (length 1-4 monomers) showed moderate self-quenching except xC, which showed enhancement of Phifl with increasing length. Interestingly, multimers of xA emitted at longer wavelengths (520 nm) as an apparent excimer. Hybridization of an oligonucleotide to the DNA adjacent to the xDNA bases (with the xDNA portion overhanging) resulted in no change in fluorescence. However, addition of one, two, or more DNA bases in these duplexes opposite the xDNA portion resulted in a number of significant fluorescence responses, including wavelength shifts, enhancements, or quenching. The strongest responses were the enhancement of (xG)n emission by hybridization of one or more adenines opposite them, and the quenching of (xT)n and (xC)n emission by guanines opposite. The data suggest multiple ways in which the xDNA bases, both alone and in oligomers, may be useful as tools in biophysical analysis and biotechnological applications.

    View details for DOI 10.1021/ja0782347

    View details for Web of Science ID 000254173600060

    View details for PubMedID 18311973

  • Quenching of fluorescent nucleobases by neighboring DNA: The "Insulator" concept CHEMBIOCHEM Wilson, J. N., Cho, Y., Tan, S., Cuppoletti, A., Kool, E. T. 2008; 9 (2): 279-285


    Fluorescent nucleosides are widely used as probes of biomolecular structure and mechanism in the context of DNA, but they often exhibit low quantum yields because of quenching by neighboring DNA bases. Here we characterize the quenching by DNA of fluorescent nucleosides that have pyrene (Y), perylene (E), benzopyrene (B), or 2-aminopurine (2AP) as nucleobase replacements, and we investigate the effect of inserting varied nucleosides as potential "insulators" between the fluorescent nucleosides and other nearby DNA bases as a strategy for increasing quantum yields. The data show that the hydrocarbons are quenched by adjacent pyrimidines, with thymine being the strongest quencher. The quantum yield of pyrene is quenched 120-fold by a single adjacent T, that of benzopyrene tenfold, and that of perylene by a factor of 2.5. Quenching of excimer and exciplex dinucleoside labels (Y-Y, Y-E, E-E, etc.) was considerably lessened, but was strongest with neighboring thymine. 2-Aminopurine (2AP) is most strongly quenched (15-fold) by neighboring G. We tested four different insulator candidates for reducing this quenching by measuring the fluorescence of short oligonucleotides containing insulators placed between a fluorescent base and a quenching base. The insulators tested were a C(3) abasic spacer (S), dihydrothymidine nucleoside (DHT), terphenyl nucleoside (TP), and adenine deoxynucleoside (dA). Results showed that the abasic spacer had little effect on quenching, while the other three had substantial effects. DHT and terphenyl enhanced fluorescence of the fluorophores by factors of 5 to 70. Adenine base reduced the quenching of pyrene 40-fold. The results underscore the importance of the nearest neighbors in DNA-quenching mechanisms, and establish simple strategies for enhancing fluorescence in labeled DNAs.

    View details for DOI 10.1002/cbic.200700381

    View details for Web of Science ID 000252880000015

    View details for PubMedID 18072185

  • Quenched autoligation probes. Methods in molecular biology (Clifton, N.J.) Silverman, A. P., Abe, H., Kool, E. T. 2008; 429: 161-170


    Methods are described for preparation and use of quenched autoligation (QUAL) probes. These modified oligonucleotide fluorescent probes can be used to detect DNA and RNA in solution, on solid surfaces, and in fixed and living bacterial and human cells. They are quenched probes, and thus provide a "lighting up" signal in a single step, without removing unbound or unreacted probes from the analyte. QUAL probe signals can be detected by fluorescence spectrometer, fluorescence microscope, or flow cytometry. These probes can distinguish between very small variations, including single nucleotide differences, in nucleic acid targets. The described method includes a description of how to prepare the needed dabsyl quencher linker, how to prepare the QUAL probes by DNA synthesizer, and how to employ them in detecting nucleic acids in solution and in detecting RNAs in bacterial and human cells.

    View details for DOI 10.1007/978-1-60327-040-3_11

    View details for PubMedID 18695965

  • Studies of oligodeoxyfluorosides (ODFs) as FRET probes for DNA hybridization. Nucleic acids symposium series (2004) Teo, Y. N., Kool, E. T. 2008: 233-234


    Oligodeoxyfluorosides (ODFs) are a novel system of stacked, electronically interacting fluorophores built on the DNA scaffold. Here we describe early studies of these ODFs as potential universal FRET donors and as reporters of DNA hybridization.

    View details for DOI 10.1093/nass/nrn118

    View details for PubMedID 18776339

  • Towards the replication of xDNA, a size-expanded unnatural genetic system. Nucleic acids symposium series (2004) Krueger, A. T., Lu, H., Højland, T., Liu, H., Gao, J., Kool, E. T. 2008: 455-456


    Here we study the viability of an unnatural genetic system with size-expanded geometry (xDNA). xDNA contains base pairs 2.4 A larger than those of natural DNA. The expanded geometry is expected to be problematic for the natural high-fidelity replication machinery required to process genetic information. However, initial studies with a variety of DNA polymerases are promising in demonstrating replication of these unnatural bases. The results suggest the future possible viability of fully functional unnatural genetic systems, and give insight into the steric limits of some natural DNA polymerases.

    View details for DOI 10.1093/nass/nrn231

    View details for PubMedID 18776450

  • Steric effects in RNA interference: Probing the influence of nucleobase size and shape CHEMISTRY-A EUROPEAN JOURNAL Somoza, A., Silverman, A. P., Miller, R. M., Chelliserrykattil, J., Kool, E. T. 2008; 14 (26): 7978-7987


    Nonpolar nucleosides with varying size and shape have been used to study the hydrogen-bonding stabilization and steric effects on RNA interference. The uracil and adenine residues of siRNA guide strands have been replaced by nonpolar isosteres of uracil and adenine and by steric variants. RNAi experiments targeting Renilla luciferase mRNA have shown close correlation between siRNA thermal stability and gene suppression. Interestingly, siRNA modified at position 7 on the guide strand does not follow this correlation, having substantial RNAi activity despite low thermal stability. Sequence-selectivity studies were carried out at this position with mutated target mRNAs and nucleobase analogues with varied size (2,4-difluoro- and 2,4-dichlorobenzene) and different shape (2,3-dichlorobenzene, 4-methylbenzimidazole). The results point out the importance of nucleobase shape and steric effects in RNA interference.

    View details for DOI 10.1002/chem.200800837

    View details for Web of Science ID 000259523500028

    View details for PubMedID 18624291

  • New, stronger nucleophiles for nucleic acid-templated chemistry: Synthesis and application in fluorescence detection of cellular RNA BIOORGANIC & MEDICINAL CHEMISTRY Miller, G. P., Silverman, A. P., Kool, E. T. 2008; 16 (1): 56-64


    Nucleic acid-templated chemistry is a promising strategy for imaging genetic sequences in living cells. Here we describe the synthesis of two new nucleophiles for use in templated nucleophilic displacements with DNA probes. The nucleophilic groups are phosphorodithioate and phosphorotrithioate; we report on synthetic methods for introducing these groups at the 3'-terminus of oligonucleotides. Both new nucleophiles are found to be more highly reactive than earlier phosphoromonothioates. This increased nucleophilicity is shown to result in more rapid templated reactions with electrophilic DNA probes. The new probes were demonstrated in detection of specific genetic sequences in solution, with clear signal over background being generated in as little as 20 min. The probes were also tested for imaging ribosomal RNA sequences in live Escherichia coli; useful signal was generated in 20 min to 1h, approximately one quarter to one-half the time of earlier monothioate probes, and the signal-to-noise ratio was increased as well.

    View details for DOI 10.1016/j.bmc.2007.04.051

    View details for Web of Science ID 000253345400007

    View details for PubMedID 17502150

  • Efficient quenching of oligomeric fluorophores on a DNA backbone JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Wilson, J. N., Teo, Y. N., Kool, E. T. 2007; 129 (50): 15426-?

    View details for DOI 10.1021/Ja075968a

    View details for Web of Science ID 000251581900008

    View details for PubMedID 18027944

  • Steric and electrostatic effects in DNA synthesis by the SOS-induced DNA polymerases II and IV of Escherichia coli BIOCHEMISTRY Silverman, A. P., Jiang, Q., Goodman, M. F., Kool, E. T. 2007; 46 (48): 13874-13881


    The SOS-induced DNA polymerases II and IV (pol II and pol IV, respectively) of Escherichia coli play important roles in processing lesions that occur in genomic DNA. Here we study how electrostatic and steric effects play different roles in influencing the efficiency and fidelity of DNA synthesis by these two enzymes. These effects were probed by the use of nonpolar shape analogues of thymidine, in which substituted toluenes replace the polar thymine base. We compared thymine with nonpolar analogues to evaluate the importance of hydrogen bonding in the polymerase active sites, while we used comparisons among a set of variably sized thymine analogues to measure the role of steric effects in the two enzymes. Steady-state kinetics measurements were carried out to evaluate activities for nucleotide insertion and extension. The results showed that both enzymes inserted nucleotides opposite nonpolar template bases with moderate to low efficiency, suggesting that both polymerases benefit from hydrogen bonding or other electrostatic effects involving the template base. Surprisingly, however, pol II inserted nonpolar nucleotide (dNTP) analogues into a primer strand with high (wild-type) efficiency, while pol IV handled them with an extremely low efficiency. Base pair extension studies showed that both enzymes bypass non-hydrogen-bonding template bases with moderately low efficiency, suggesting a possible beneficial role of minor groove hydrogen bonding interactions at the N-1 position. Measurement of the two polymerases' sensitivity to steric size changes showed that both enzymes were relatively flexible, yielding only small kinetic differences with increases or decreases in nucleotide size. Comparisons are made to recent data for DNA pol I (Klenow fragment), the archaeal polymerase Dpo4, and human pol kappa.

    View details for DOI 10.1021/bi700851z

    View details for Web of Science ID 000251241500021

    View details for PubMedID 17988102

  • Model systems for understanding DNA base pairing CURRENT OPINION IN CHEMICAL BIOLOGY Krueger, A. T., Kool, E. T. 2007; 11 (6): 588-594


    The fact that nucleic acid bases recognize each other to form pairs is a canonical part of the dogma of biology. However, they do not recognize each other well enough in water to account for the selectivity and efficiency that is needed in the transmission of biological information through a cell. Thus proteins assist in this recognition in multiple ways, and recent data suggest that these mechanisms of recognition can vary widely with context. To probe how the chemical differences of the four nucleobases are defined in various biological contexts, chemists and biochemists have developed modified versions that differ in their polarity, shape, size, and functional groups. This brief review covers recent advances in this field of research.

    View details for DOI 10.1016/j.cbpa.2007.09.019

    View details for Web of Science ID 000251954100003

    View details for PubMedID 17967435

  • RNA probes of steric effects in active sites: High flexibility of HIV-1 reverse transcriptase JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Silverman, A. P., Kool, E. T. 2007; 129 (35): 10626-?

    View details for DOI 10.1021/ja072791b

    View details for Web of Science ID 000249208300006

    View details for PubMedID 17696348

  • Nonpolar isosteres of damaged DNA bases: Effective mimicry of mutagenic properties of 8-oxopurines JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Taniguchi, Y., Kool, E. T. 2007; 129 (28): 8836-8844


    A substantial fraction of mutations that arise in the cell comes from oxidative damage to DNA bases. Oxidation of purine bases at the 8-position, yielding 8-oxo-G and 8-oxo-A, results in conformational changes (from anti to syn) that cause miscoding during DNA replication. Here we describe the synthesis and biophysical and biochemical properties of low-polarity shape mimics of 8-oxopurines, and we report that these new analogues exhibit remarkable mimicry of the mutagenic properties of the natural damaged bases. A 2-chloro-4-fluoroindole nucleoside (1) was designed as an isosteric analogue of 8-oxo-dG, and a 2-chloro-4-methylbenzimidazole nucleoside (2) as a mimic of 8-oxo-dA. The nucleosides were prepared by reaction of the parent heterocycles with Hoffer's chlorodeoxyribose derivative. Structural studies of the free nucleosides 1 and 2 revealed that both bases are oriented syn, thus mimicking the conformation of the oxopurine nucleosides. Suitably protected phosphoramidite derivatives were prepared for incorporation into synthetic DNAs, to be used as probes of DNA damage responses, and 5'-triphosphate derivatives (3 and 4) were synthesized as analogues of damaged nucleotides in the cellular nucleotide pool. Base pairing studies in 12-mer duplexes showed that 1 and 2 have low affinity for polar pairing partners, consistent with previous nonpolar DNA base analogues. However, both compounds pair with small but significant selectivity for purine partners, consistent with the idea that the syn purine geometry leads to pyrimidine-like shapes. Steady-state kinetics studies of 1 and 2 were carried out with the Klenow fragment of Escherichia coli DNA Pol I (exo-) in single-nucleotide insertions. In the DNA template, the analogues successfully mimicked the mutagenic behavior of oxopurines, with 1 being paired selectively with adenine and 2 pairing selectively with guanine. The compounds showed similar mutagenic behavior as nucleoside triphosphate analogues, being preferentially inserted opposite mutagenic purine partners. The results suggest that much of the mutagenicity of oxopurines arises from their shapes in the syn conformation rather than from electrostatic and hydrogen-bonding effects. The new analogues are expected to be generally useful as mechanistic probes of cellular responses to DNA damage.

    View details for DOI 10.1021/ja071970q

    View details for Web of Science ID 000247966200038

    View details for PubMedID 17592846

  • Oligodeoxyfluorosides: strong sequence dependence of fluorescence emission TETRAHEDRON Wilson, J. N., Gao, J., Kool, E. T. 2007; 63 (17): 3427-3433


    We describe the properties of a series of oligomeric polyfluorophores assembled on the DNA backbone. The eleven oligomers (oligodeoxyfluorosides, ODFs), 4 to 7 monomers in length, were composed of only two fluorescent monomers and a spacer in va ried sequences, and were designed to test how fluorescent nucleobases can interact electronically to yield complexity in fluorescence emission. The monomer fluorophores were deoxyribosides of pyrene and perylene, which emit light in violet and blue wavele ngths respectively. The experiments show that simple variation of sequence and spacing can dramatically change fluorescence, yielding emission maxima ranging from 380 to 557 nm and visible colors from violet to orange-red. Fluorescence lifetimes data, excitation spectra, and absorption data point to a number of multi-fluorophore electronic interactions, including pyrene-pyrene and perylene-perylene excimers, pyrene-perylene exciplexes, as well as monomer dye emissions, contributing to the final spectral outcomes. Thus, two simple fluorophores can be readily combined to give emissions over much of the visible spectrum, all requiring only a single excitation. The results demonstrate that fluorescent nucleobases in oligomeric form can act cooperatively as electronic units, and that fluorophore sequence in such oligomers is as important as fluorophore composition in determining fluorescence properties.

    View details for DOI 10.1016/j.tet.2006.07.113

    View details for Web of Science ID 000248294800002

  • Synthesis and properties of size-expanded DNAs: Toward designed, functional genetic systems ACCOUNTS OF CHEMICAL RESEARCH Krueger, A. T., Lu, H., Lee, A. H., Kool, E. T. 2007; 40 (2): 141-150


    We describe the design, synthesis, and properties of DNA-like molecules in which the base pairs are expanded by benzo homologation. The resulting size-expanded genetic helices are called xDNA ("expanded DNA") and yDNA ("wide DNA"). The large component bases are fluorescent, and they display high stacking affinity. When singly substituted into natural DNA, they are destabilizing because the benzo-expanded base pair size is too large for the natural helix. However, when all base pairs are expanded, xDNA and yDNA form highly stable, sequence-selective double helices. The size-expanded DNAs are candidates for components of new, functioning genetic systems. In addition, the fluorescence of expanded DNA bases makes them potentially useful in probing nucleic acids.

    View details for DOI 10.1021/ar068200o

    View details for Web of Science ID 000244248000005

    View details for PubMedID 17309194

  • The model student: what chemical model systems can teach us about biology NATURE CHEMICAL BIOLOGY Kool, E. T., Waters, M. L. 2007; 3 (2): 70-73


    Model systems have evolved with the times, making use of modern biological methods and incorporating biological complexity. This evolution has increased the relevance of models as tools for studying biology.

    View details for DOI 10.1038/nchembio0207-70

    View details for Web of Science ID 000243685800002

    View details for PubMedID 17235337

  • Site-directed mutagenesis in the fingers subdomain of HIV-1 reverse transcriptase reveals a specific role for the beta 3-beta 4 hairpin loop in dNTP selection JOURNAL OF MOLECULAR BIOLOGY Garforth, S. J., Kim, T. W., Parniak, M. A., Kool, E. T., Prasad, V. R. 2007; 365 (1): 38-49


    HIV-1 reverse transcriptase shares the key features of high fidelity polymerases, such as a closed architecture of the active site, but displays a level of fidelity that is intermediate to that of high fidelity, replicative polymerases and low fidelity translesion synthesis (TLS) polymerases. The beta3-beta4 loop of the HIV-1 RT fingers subdomain makes transient contacts with the dNTP and template base. To investigate the role of active site architecture in HIV-1 RT fidelity, we truncated the beta3-beta4 loop, eliminating contact between Lys65 and the gamma-phosphate of dNTP. The mutant, in a manner reminiscent of TLS polymerases, was only able to incorporate a nucleotide that was capable of base-pairing with the template nucleotide, but not a nucleotide shape-analog incapable of Watson-Crick hydrogen bonding. Unexpectedly, however, the deletion mutant differed from the TLS polymerases in that it displayed an increased fidelity. The increased fidelity was associated with reduced dNTP binding affinity as measured using the dead end complex formation. In an effort to delineate the specific amino acid residue in the deleted segment responsible for this phenotype, we examined the K65 residue. Two substitution mutants, K65R and K65A were studied. The K65A mutant behaved similarly to the deletion mutant displaying dependence on Watson-Crick hydrogen bonding, increased fidelity and reduced dNTP-binding, while the K65R was more akin to wild-type enzyme. These results underscore the key role of the K65 residue in the phenotype observed in the deletion mutant. Based on the well-known electrostatic interaction between K65 and the gamma-phosphate moiety of incoming dNTP substrate in the ternary complex structure of HIV-1 RT, we conclude that non-discriminatory interactions between beta3-beta4 loop and the dNTP in wild-type HIV-1 RT help lower dNTP selectivity. Our results show that the fidelity of dNTP insertion is influenced by protein interactions with the triphosphate moiety.

    View details for DOI 10.1016/j.jmb.2006.09.057

    View details for Web of Science ID 000243199300005

    View details for PubMedID 17055529

  • Syntheses and properties of low-polarity shape mimics of 8-oxopurines. Nucleic acids symposium series (2004) Taniguchi, Y., Kool, E. T. 2007: 217-218


    A number of mutations that arise in the cell come from oxidative damage to DNA bases. Oxidation of purine bases at the 8-position, yielding 8-oxoguanosine and 8-oxoadenosine, results in conformation changes that cause miscoding during DNA replication. For example, 8-oxodG in the syn conformation is complementary to adenine in the hydrogen bonding. Here, we report the synthesis, biophysical and biochemical properties of low-polarity shape mimics of 8-oxopurines. A 2-chloro-4-fluoroindole (1) nucleoside was designed as a mimic of 8-oxodG, and a 2-chloro-4-methylbenzimidazole nucleoside (2) as a mimic of 8-oxodA. Structural studies of the free nucleoside revealed that both bases are preferred syn conformation, thus mimicking the conformation of the oxopurine nucleosides. Base pairing studies showed that both compounds in the oligonucleotides pair with selectivity for purine partners. In the DNA template containing new oxopurine analogs, dATP and dGTP were incorporated opposite 1 and 2 respectively by the Klenow. Moreover, 5'-triphosphate derivatives (3 and 4) were incorporated into DNA opposite purine nucleobase. Thus the new analogs are expected to be useful as mechanistic probes cellular responses to DNA oxidative damage.

    View details for PubMedID 18029664

  • Oligonucleotide probes for RNA-targeted fluorescence in situ hybridization ADVANCES IN CLINICAL CHEMISTRY, VOL 43 Silverman, A. P., Kool, E. T. 2007; 43: 79-115


    The need for accurate and rapid methodology for detecting cells in environmental and clinical samples has led to the development of in situ detection methods, where fixed or intact cells can be imaged directly. In this chapter, we focus on the use of labeled oligonucleotide probes in fluorescence in situ hybridization (FISH). We give an overview of FISH probe design, covering issues of affinity and specificity of probes, probe backbone options, cellular targets, and accessibility of target sequences. Decisions that must be made to design optimal probes are evaluated, and available resources to assist in probe design, such as secondary structure, Tm calculation, and site accessibility software, are discussed. We cover different types of FISH probes that have been reported in the recent literature, including standard fluorescently labeled oligonucleotide probes and newer classes of quenched oligonucleotide probes: molecular beacons and quenched autoligation probes. Advantages and disadvantages of the different probe types are examined and recent literature applications are discussed. The current state of the art in the field as well as limitations and challenges in detection are evaluated.

    View details for DOI 10.1016/S0065-2423(06)43003-1

    View details for Web of Science ID 000243457200003

    View details for PubMedID 17249381

  • RNA-templated chemistry in cells: Discrimination of Escherichia, Shigella and Salmonella bacterial strains with a new two-color FRET strategy CHEMBIOCHEM Silverman, A. P., Baron, E. J., Kool, E. T. 2006; 7 (12): 1890-1894

    View details for DOI 10.1002/cbic.200600278

    View details for Web of Science ID 000242981500012

    View details for PubMedID 17031884

  • Nonpolar nucleobase analogs illuminate requirements for site-specific DNA cleavage by vaccinia topoisomerase JOURNAL OF BIOLOGICAL CHEMISTRY Yakovleva, L., Lai, J., Kool, E. T., Shuman, S. 2006; 281 (47): 35914-35921


    Vaccinia DNA topoisomerase forms a covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate at a specific target site 5'-C(+5)C(+4)C(+3)T(+2)T(+1)p downward arrow N(-1) in duplex DNA. Here we study the effects of nonpolar pyrimidine isosteres difluorotoluene (F) and monofluorotoluene (D) and the nonpolar purine analog indole at individual positions of the scissile and nonscissile strands on the rate of single-turnover DNA transesterification and the cleavage-religation equilibrium. Comparison of the effects of nonpolar base substitution to the effects of abasic lesions reported previously allowed us to surmise the relative contributions of base-stacking and polar edge interactions to the DNA transesterification reactions. For example, the deleterious effects of eliminating the +2T base on the scissile strand were rectified by introducing the nonpolar F isostere, whereas the requirement for the +1T base was not elided by F substitution. We impute a role for +1T in recruiting the catalytic residue Lys-167 to the active site. Topoisomerase is especially sensitive to suppression of DNA cleavage upon elimination of the +4G and +3G bases of the nonscissile strand. Indole provided little or no gain of function relative to abasic lesions. Inosine substitutions for +4G and +3G had no effect on transesterification rate, implying that the guanine exocyclic amine is not a critical determinant of DNA cleavage. Prior studies of 2-aminopurine and 7-deazaguanine effects had shown that the O6 and N7 of guanine were also not critical. These findings suggest that either the topoisomerase makes functionally redundant contacts with polar atoms (likely via Tyr-136, a residue important for precleavage active site assembly) or that it relies on contacts to N1 or N3 of the purine ring. The cleavage-religation equilibrium is strongly skewed toward trapping of the covalent intermediate by elimination of the +1A base of the nonscissile strand; the reaction equilibrium is restored by +1 indole, signifying that base stacking flanking the nick is critical for the religation step. Our findings highlight base isosteres as valuable tools for the analysis of proteins that act on DNA in a site-specific manner.

    View details for DOI 10.1074/jbc.M608349200

    View details for Web of Science ID 000242100500038

    View details for PubMedID 17005552

  • Toward a designed, functioning genetic system with expanded-size base pairs: Solution structure of the eight-base xDNA double helix JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Lynch, S. R., Liu, H., Gao, J., Kool, E. T. 2006; 128 (45): 14704-14711


    We describe the NMR-derived solution structure of the double-helical form of a designed eight-base genetic pairing system, termed xDNA. The benzo-homologous xDNA design contains base pairs that are wider than natural DNA pairs by ca. 2.4 A (the width of a benzene ring). The eight component bases of this xDNA helix are A, C, G, T, xA, xT, xC, and xG. The structure was solved in aqueous buffer using 1D and 2D NMR methods combined with restrained molecular dynamics. The data show that the decamer duplex is right-handed and antiparallel, and hydrogen-bonded in a way analogous to that of Watson-Crick DNA. The sugar-phosphate backbone adopts a regular conformation similar to that of B-form DNA, with small dihedral adjustments due to the larger circumference of the helix. The grooves are much wider and more shallow than those of B-form DNA, and the helix turn is slower, with ca. 12 base pairs per 360 degrees turn. There is an extensive intra- and interstrand base stacking surface area, providing an explanation for the greater stability of xDNA relative to natural DNA. There is also evidence for greater motion in this structure compared to a previous two-base-expanded helix; possible chemical and structural reasons for this are discussed. The results confirm paired self-assembly of the designed xDNA system. This suggests the possibility that other genetic system structures besides the natural one might be functional in encoding information and transferring it to new complementary strands.

    View details for DOI 10.1021/ja065606n

    View details for Web of Science ID 000241857200060

    View details for PubMedID 17090058

  • Detecting RNA and DNA with templated chemical reactions CHEMICAL REVIEWS Silverman, A. P., Kool, E. T. 2006; 106 (9): 3775-3789

    View details for DOI 10.1021/cr050057+

    View details for Web of Science ID 000240463400007

    View details for PubMedID 16967920

  • Exploring the limits of DNA size: Naphtho-homologated DNA bases and pairs JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Lee, A. H., Kool, E. T. 2006; 128 (28): 9219-9230


    A new design for DNA bases and base pairs is described in which the pyrimidine bases are widened by naphtho-homologation. Two naphtho-homologated deoxyribosides, dyyT (1) and dyyC (2), were synthesized and could be incorporated into oligonucleotides as suitably protected phosphoramidite derivatives. The deoxyribosides were found to be fluorescent, with emission maxima at 446 and 433 nm, respectively. Studies with single substitutions of 1 and 2 in the natural DNA context revealed exceptionally strong base stacking propensity for both. Sequences containing multiple substitutions of 1 and 2 paired opposite adenine and guanine were subsequently mixed and studied by several analytical methods. Data from UV mixing experiments, FRET measurements, fluorescence quenching experiments, and hybridizations on beads suggest that complementary "doublewide DNA" (yyDNA) strands may self-assemble into helical complexes with 1:1 stoichiometry. Data from thermal denaturation plots and CD spectra were less conclusive. Control experiments in one sequence context gave evidence that yyDNA helices, if formed, are preferentially antiparallel and are sequence selective. Hypothesized base pairing schemes are analogous to Watson-Crick pairing, but with glycosidic C1'-C1' distances widened by over 45%, to ca. 15.2 A. The possible self-assembly of the double-wide DNA helix establishes a new limit for the size of information-encoding, DNA-like molecules, and the fluorescence of yyDNA bases suggests uses as reporters in monomeric and oligomeric forms.

    View details for DOI 10.1021/ja0619004

    View details for Web of Science ID 000238972900052

    View details for PubMedID 16834396

  • Sensing metal ions with DNA building blocks: Fluorescent pyridobenzimidazole nucleosides JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Kim, S. J., Kool, E. T. 2006; 128 (18): 6164-6171


    We describe novel fluorescent N-deoxyribosides (1 and 2) having 2-pyrido-2-benzimidazole and 2-quino-2-benzimidazole as aglycones. The compounds were prepared from the previously unknown heterocyclic precursors and Hoffer's chlorosugar, yielding alpha anomers as the chief products. X-ray crystal structures confirmed the geometry and showed that the pyridine and benzimidazole ring systems deviated from coplanarity in the solid state by 154 degrees and 140 degrees , respectively. In methanol compounds 1 and 2 had absorption maxima at 360 and 370 nm, respectively, and emission maxima at 494 and 539 nm. Experiments revealed varied fluorescence responses of the nucleosides to a panel of 17 monovalent, divalent, and trivalent metal ions in methanol. One or both of the nucleosides showed significant changes with 10 of the metal ions. The most pronounced spectral changes for ligand-nucleoside 1 included red shifts in fluorescence (Au(+), Au(3+)), strong quenching (Cu(2+), Ni(2+), Pt(2+)), and substantial enhancements in emission intensity coupled with red shifts (Ag(+), Cd(2+), Zn(2+)). The greatest spectral changes for ligand-nucleoside 2 included a red shift in fluorescence (Ag(+)), a blue shift (Cd(2+)), strong quenching (Pd(2+), Pt(2+)), and substantial enhancements in emission intensity coupled with a blue shift (Zn(2+)). The compounds could be readily incorporated into oligodeoxynucleotides, where an initial study revealed that they retained sensitivity to metal ions in aqueous solution and demonstrated possible cooperative sensing behavior with several ions. The two free nucleosides alone can act as differential sensors for multiple metal ions, and they are potentially useful monomers for contributing metal ion sensing capability to DNAs.

    View details for DOI 10.1021/ja0581806

    View details for Web of Science ID 000237468900050

    View details for PubMedID 16669686

  • Enzymatic synthesis of fluorescent oligomers assembled on a DNA backbone CHEMBIOCHEM Cho, Y. J., Kool, E. T. 2006; 7 (4): 669-672


    A number of research laboratories have investigated the properties of multichromophore molecules and their applications in materials science and in biotechnology. Previous approaches for preparing such molecules have involved traditional organic synthesis. Here we describe the one-step enzymatic synthesis of such a multichromophore species by using a DNA-polymerizing enzyme (terminal deoxynucleotidyl transferase (TdT)). We find that a nucleotide-like molecule with pyrene replacing the DNA base (dPTP) can be accepted as a substrate for this enzyme to produce discrete chromophores that have 3 or 4 pyrenes consecutively, depending on which anomer (alpha or beta) is used. Products were characterized by gel electrophoresis, mass spectrometry, and fluorescence. The reaction was found to change the fluorescence emission of the chromophore from a maximum at 375 nm (the monomer nucleotide) to 490 nm in the oligomeric product. This new green-white emission is consistent with the formation of a pyrene excimer between adjacent pyrene glycosides, which exhibit a large Stokes shift of 130 nm. The enzymatic synthesis of the pyrene excimer might have applications in homogeneous biological assays for DNA fragments, such as those that arise during apoptosis.

    View details for DOI 10.1002/cbic.200500515

    View details for Web of Science ID 000236685900016

    View details for PubMedID 16502476

  • Varying DNA base-pair size in subangstrom increments: Evidence for a loose, not large, active site in low-fidelity Dpo4 polymerase BIOCHEMISTRY Mizukami, S., Kim, T. W., Helquist, S. A., Kool, E. T. 2006; 45 (9): 2772-2778


    We describe the first systematic test of steric effects in the active site of a Y-family DNA polymerase, Dpo4. It has been hypothesized that low-fidelity repair polymerases in this family more readily accept damaged or mismatched base pairs because of a sterically more open active site, which might place lower geometric constraints on the incipient pair. We have tested the origin of low fidelity by use of five nonpolar thymidine analogues that vary in size by a total of 1.0 A over the series. The efficiency and fidelity of base-pair synthesis was measured by steady-state kinetics for single-nucleotide insertions. Analogues were examined both as incoming deoxynucleoside triphosphate (dNTP) derivatives and as template bases. The results showed that Dpo4 preferred to pair the thymidine shape mimics with adenine and, surprisingly, the preferred size was at the center of the range, the same optimum size as recently found for the high-fidelity Klenow fragment (Kf) of Escherichia coli DNA Pol I. However, the size preference with Dpo4 was quite small, varying by a factor of only 30-35 from most to least efficient thymidine analogue. This is in marked contrast to Kf, which showed a rigid size preference, varying by 1100-fold from best to worst. The fidelity for the non-hydrogen-bonding analogues in pairing with A over T, C, or G was much lower in Dpo4 than in the previous high-fidelity enzyme. The data establish that, unlike Kf, Dpo4 has very low steric selectivity and that steric effects alone cannot explain the fidelity (albeit low) that Dpo4 has for a correct base pair; the findings suggest that hydrogen bonds may be important in determining the fidelity of this enzyme. The results suggest that the low steric selectivity of this enzyme is the result of a conformationally flexible or loose active site that adapts with small energetic cost to different base-pair sizes (as measured by the glycosidic C1'-C1' distance), rather than a spatially large active site.

    View details for DOI 10.1021/bi051961z

    View details for Web of Science ID 000235792500003

    View details for PubMedID 16503632

  • Dynamics of nucleotide incorporation: Snapshots revealed by 2-ammopurine fluorescence studies BIOCHEMISTRY Hariharan, C., Bloom, L. B., Helquist, S. A., Kool, E. T., Reha-Krantz, L. J. 2006; 45 (9): 2836-2844


    Formation of a noncanonical base pair between dFTP, a dTTP analogue that cannot form H bonds, and the fluorescent base analogue 2-aminopurine (2AP) was studied in order to discover how the bacteriophage T4 DNA polymerase selects nucleotides with high accuracy. Changes in 2AP fluorescence intensity provided a spectroscopic reporter of the nucleotide binding reactions, which were combined with rapid-quench, pre-steady-state reactions to measure product formation. These studies supported and extended previous findings that the T4 DNA polymerase binds nucleotides in multiple steps with increasing selectivity. With 2AP in the template position, initial dTTP binding was rapid but selective: K(d(dTTP)) (first step) = 31 microM; K(d(dCTP)) (first step) approximately 3 mM. In studies with dFTP, this step was revealed to have two components: formation of an initial preinsertion complex in which H bonds between bases in the newly forming base pair were not essential, which was followed by formation of a final preinsertion complex in which H bonds assisted. The second nucleotide binding step was characterized by increased discrimination against dTTP binding opposite template 2AP, K(d) (second step) = 367 microM, and additional conformational changes were detected in ternary enzyme-DNA-dTTP complexes, as expected for forming closed complexes. We demonstrate here that the second binding step occurs before formation of the phosphodiester bond. Thus, the high fidelity of nucleotide insertion by T4 DNA polymerase is accomplished by the sequential application of selectivity in first forming accurate preinsertion complexes, and then additional conformational changes are applied that further increase discrimination against incorrect nucleotides.

    View details for DOI 10.1021/bi051644s

    View details for Web of Science ID 000235792500009

    View details for PubMedID 16503638

  • Functional evidence for a small and rigid active site in a high fidelity DNA polymerase - Probing T7 DNA polymerase with variably sized base pairs JOURNAL OF BIOLOGICAL CHEMISTRY Kim, T. W., Brieba, L. G., Ellenberger, T., Kool, E. T. 2006; 281 (4): 2289-2295


    Hypotheses on the origins of high fidelity in replicative DNA polymerases have recently focused on the importance of geometric or steric effects in this selectivity. Here we reported a systematic study of the effects of base pair size in T7 DNA polymerase (pol), the replicative enzyme for bacteriophage T7. We varied base pair size in very small (0.25 A) increments by use of a series of nonpolar thymidine shape mimics having gradually increasing size. Steady-state kinetics were evaluated for the 5A7A exonuclease-deficient mutant in a 1:1 complex with thioredoxin. For T7 pol, we studied insertion of natural nucleotides opposite variably sized T analogs in the template and, conversely, for variably sized dTTP analogs opposite natural template bases. The enzyme displayed extremely high selectivity for a specific base pair size, with drops in efficiency of as much as 280-fold for increases of 0.4 A beyond an optimum size approximating the size of a natural pair. The enzyme also strongly rejected pairs that were smaller than the optimum by as little as 0.3 A. The size preferences with T7 DNA pol were generally smaller, and the steric rejection was greater than DNA pol I Klenow fragment, correlating with the higher fidelity of the former. The hypothetical effects of varied active site size and rigidity are discussed. The data lend direct support to the concept that active site tightness is a chief determinant of high fidelity of replicative polymerases and that a less rigid (looser) and larger active site can lead to lower fidelity.

    View details for DOI 10.1074/jbc.M510744200

    View details for Web of Science ID 000234760400054

    View details for PubMedID 16311403

  • DNA polymerase catalysis in the absence of Watson-Crick hydrogen bonds: Analysis by single-turnover kinetics BIOCHEMISTRY Potapova, O., Chan, C., DeLucia, A. M., Helquist, S. A., Kool, E. T., Grindley, N. D., Joyce, C. M. 2006; 45 (3): 890-898


    We report the first pre-steady-state kinetic studies of DNA replication in the absence of hydrogen bonds. We have used nonpolar nucleotide analogues that mimic the shape of a Watson-Crick base pair to investigate the kinetic consequences of a lack of hydrogen bonds in the polymerase reaction catalyzed by the Klenow fragment of DNA polymerase I from Escherichia coli. With a thymine isostere lacking hydrogen-bonding ability in the nascent pair, the efficiency (k(pol)/Kd) of the polymerase reaction is decreased by 30-fold, affecting the ground state (Kd) and transition state (k(pol)) approximately equally. When both thymine and adenine analogues in the nascent pair lack hydrogen-bonding ability, the efficiency of the polymerase reaction is decreased by about 1000-fold, with most of the decrease attributable to the transition state. Reactions using nonpolar analogues at the primer-terminal base pair demonstrated the requirement for a hydrogen bond between the polymerase and the minor groove of the primer-terminal base. The R668A mutation of Klenow fragment abolished this requirement, identifying R668 as the probable hydrogen-bond donor. Detailed examination of the kinetic data suggested that Klenow fragment has an extremely low tolerance of even minor deviations of the analogue base pairs from ideal Watson-Crick geometry. Consistent with this idea, some analogue pairings were better tolerated by Klenow fragment mutants having more spacious active sites. In contrast, the Y-family polymerase Dbh was much less sensitive to changes in base pair dimensions and more dependent upon hydrogen bonding between base-paired partners.

    View details for DOI 10.1021/bi051792i

    View details for Web of Science ID 000234988600025

    View details for PubMedID 16411765

  • Enhanced base pairing and replication efficiency of thiothymidines, expanded-size variants of thymidine JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Sintim, H. O., Kool, E. T. 2006; 128 (2): 396-397


    Here we test the steric effects of added size on DNA base pairing and replication by the use of thiocarbonyl group replacements for natural nucleoside thymidine. The 2-thioT (2S) and 4-thioT (4S) nucleosides were reported previously, but their pairing specificity and replication fidelity were unknown. We find that 2S pairs with higher specificity than thymidine, and both 2S and 4S nucleoside triphosphates are replicated with higher efficiency than natural dTTP. The results indicate possible biotechnological uses for these analogues and have implications in the ongoing use of thionucleobases in cancer treatment.

    View details for DOI 10.1021/ja0562447

    View details for Web of Science ID 000234814900008

    View details for PubMedID 16402811

  • Flow cytometric detection of specific RNAs in native human cells with quenched autoligating FRET probes PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Abe, H., Kool, E. T. 2006; 103 (2): 263-268


    We describe the use of modified fluorescent-labeled oligonucleotide probes in the sequence-specific detection of messenger RNAs in live human cells. To make this detection possible, we developed a previously undescribed probe design that combines earlier quenched autoligation chemistry with a previously undescribed fluorescence resonance energy transfer (FRET) strategy to lower background signals. The probe pairs consisted of a nucleophilic 3'-phosphorothioate probe carrying a Cy5 FRET acceptor, and an electrophilic probe containing the combination of a 5' end electrophile/quencher and a fluorescein FRET donor. Probes were introduced to HL-60 cells by use of the streptolysin O pore-forming peptide. Signals from three different messenger RNAs, as well as 28S ribosomal RNA, could be detected and quantitated by flow cytometry. Probes targeted to ribosomal sequences and beta-actin mRNA also could be detected over background by confocal fluorescence microscopy. Varying the target site and probe backbone chemistry were found to have large effects on signal. The data suggest that quenched autoligating probes may be of general utility as biological tools in following localization, transcription, and processing of eukaryotic cellular messages and may have applications in diagnostic or prognostic analysis of disease-related RNAs in human tissues.

    View details for DOI 10.1073/pnas.0509938103

    View details for Web of Science ID 000234624100004

    View details for PubMedID 16384914

  • The roles of hydrogen bonding and sterics in RNA interference ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Somoza, A., Chelliserrykattil, J., Kool, E. T. 2006; 45 (30): 4994-4997

    View details for DOI 10.1002/anie.200601311

    View details for Web of Science ID 000239543300026

    View details for PubMedID 16802393

  • Fluorescent DNA base replacements: reporters and sensors for biological systems ORGANIC & BIOMOLECULAR CHEMISTRY Wilson, J. N., Kool, E. T. 2006; 4 (23): 4265-4274


    We describe the design, synthesis, and properties of nucleoside monomers in which the DNA base is replaced by fluorescent hydrocarbons and heterocycles, and the assembly of these monomers into DNA-like molecules in which the all bases are fluorescent. As monomers, such molecules have useful applications as reporters in the DNA context. The use of fluorescent DNA bases, rather than more traditional fluorophores tethered to the DNA strand, gives a more predictable location and orientation, and yields a more direct response to changes that occur within the helix. In addition to uses as monomers, such compounds can be assembled into polychromophoric oligomers ("oligodeoxyfluorosides" or ODFs). ODFs are water soluble, discrete molecules and are easily arranged into specific sequences by use of a DNA synthesizer. They have displayed a number of properties not readily available in commercial fluorophores, including large Stokes shifts, tunable excitation and emission wavelengths, and sensing responses to physical changes or molecular species in solution. We describe an approach to assembling and screening large sets of oligofluorosides for rapid identification of molecules with desirable properties. Such compounds show promise for applications in biochemistry, biology, environmental and materials applications.

    View details for DOI 10.1039/b612284c

    View details for Web of Science ID 000242005100001

    View details for PubMedID 17102869

  • The difluorotoluene debate - a decade later CHEMICAL COMMUNICATIONS Kool, E. T., Sintim, H. O. 2006: 3665-3675


    2,4-Difluorotoluene is unusual among hydrofluorocarbons because it is shaped like the DNA base thymine. It was first synthesised as a nucleotide analogue and incorporated into DNA a decade ago. Although it is a nonpolar molecule, it was found to be replicated by DNA polymerase enzymes as if it were thymine. We concluded that replication of DNA base pairs can occur without Watson-Crick hydrogen bonds, and hypothesised that steric effects, rather than these hydrogen bonds, were the main arbiters of DNA replication fidelity. A debate was initiated then, with claims by some that the molecule is polar and forms hydrogen bonds with adenine, thus supporting the hydrogen bonding theory of DNA replication. Here we discuss the evolution of this debate, and reflect on the relevant data that have since come from hundreds of papers and dozens of laboratories. Although discussion on this topic continues, the steric hypothesis for DNA replication is now widely accepted among biochemists, and the changing paradigm has been reflected in textbooks.

    View details for DOI 10.1039/b605414e

    View details for Web of Science ID 000240109400001

    View details for PubMedID 17047807

  • New designs for DNA bases: expanded DNAs and oligofluorosides. Nucleic acids symposium series (2004) Kool, E. T., Lu, H., Kim, S. J., Tan, S., Wilson, J. N., Gao, J., Liu, H. 2006: 15-16


    Properties and applications of two new classes of DNA base replacements are described. The first class is size-expanded DNA bases; these are benzo-homologous versions of A, C, T, G that yield base pairs 2.4 Angstroms larger than natural pairs. New studies have explored the ability of expanded DNA base pairs to be replicated by DNA polymerase enzymes. The second class of DNA-like molecules described here are "oligodeoxyfluorosides" (ODFs), which are deoxyribose-phosphate oligomers in which all nucleobases are replaced by hydrocarbon and heterocyclic fluorophores. These compounds have been prepared in large libraries and have been screened for unusual fluorescence and sensing properties.

    View details for PubMedID 17150794

  • Remarkable sensitivity to DNA base shape in the DNA polymerase active site ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Sintim, H. O., Kool, E. T. 2006; 45 (12): 1974-1979

    View details for DOI 10.1002/anie.200504296

    View details for Web of Science ID 000236119000036

    View details for PubMedID 16506248

  • Probing the active site tightness of DNA polymerase in subangstrom increments PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kim, T. W., Delaney, J. C., Essigmann, J. M., Kool, E. T. 2005; 102 (44): 15803-15808


    We describe the use of a series of gradually expanded thymine nucleobase analogs in probing steric effects in DNA polymerase efficiency and fidelity. In these nonpolar compounds, the base size was increased incrementally over a 1.0-A range by use of variably sized atoms (H, F, Cl, Br, and I) to replace the oxygen molecules of thymine. Kinetics studies with DNA Pol I (Klenow fragment, exonuclease-deficient) in vitro showed that replication efficiency opposite adenine increased through the series, reaching a peak at the chlorinated compound. Efficiency then dropped markedly as a steric tightness limit was apparently reached. Importantly, fidelity also followed this trend, with the fidelity maximum at dichlorotoluene, the largest compound that fits without apparent repulsion. The fidelity at this point approached that of wild-type thymine. Surprisingly, the maximum fidelity and efficiency was found at a base pair size significantly larger than the natural size. Parallel bypass and mutagenesis experiments were then carried out in vivo with a bacterial assay for replication. The cellular results were virtually the same as those seen in solution. The results provide direct evidence for the importance of a tight steric fit on DNA replication fidelity. In addition, the results suggest that even high-fidelity replicative enzymes have more steric room than necessary, possibly to allow for an evolutionarily advantageous mutation rate.

    View details for DOI 10.1073/pnas.0505113102

    View details for Web of Science ID 000233090900018

    View details for PubMedID 16249340

  • Polymerization of DNA in the absence of hydrogen bonds with terminal deoxynucleotidyl transferase Cho, Y., Helquist, S. A., Kool, E. T. AMER CHEMICAL SOC. 2005: U522-U523
  • Guanine-rich DNA nanocircles for the synthesis and characterization of long cytosine-rich telomeric DNAs CHEMBIOCHEM Hartig, J. S., Fernandez-Lopez, S., Kool, E. T. 2005; 6 (8): 1458-1462


    Short synthetic oligonucleotides derived from the human telomeric repeat have been studied recently for their ability to fold into four-stranded structures that are thought to be important to their biological function. Because telomeric DNAs are several kilobases in length, however, their folding might well be affected by cooperative or high-order interactions in these long sequences. Here, we present a new molecular system that allows for easy synthesis of very long stretches of the cytosine-rich strand of human telomeric DNA. Small circular DNAs composed of the G-rich sequence of human telomeres were prepared and used as templates in a rolling-circle replication mechanism. To facilitate the synthesis of the repetitive G-rich circles, an orthogonal base-protection strategy that made use of dimethylformamidine-protected guanine nucleobases was developed. Nanometer-scale circles ranging in size from 42 to 54 nucleotides were prepared. Subsequently, we tested the action of various DNA polymerases on these circular templates, and identified DNA Pol I (Klenow fragment) and T7 DNA polymerase as enzymes that are able to generate very long, C-rich telomeric DNA strands. Purification and initial structural examination of these C-rich polymeric products revealed evidence of a folded structure in the polymer.

    View details for DOI 10.1002/cbic.200500015

    View details for Web of Science ID 000231230000027

    View details for PubMedID 16052615

  • Evidence for a Watson-Crick hydrogen bonding requirement in DNA synthesis by human DNA polymerase kappa MOLECULAR AND CELLULAR BIOLOGY Wolfle, W. T., Washington, M. T., Kool, E. T., Spratt, T. E., Helquist, S. A., Prakash, L., Prakash, S. 2005; 25 (16): 7137-7143


    The efficiency and fidelity of nucleotide incorporation by high-fidelity replicative DNA polymerases (Pols) are governed by the geometric constraints imposed upon the nascent base pair by the active site. Consequently, these polymerases can efficiently and accurately replicate through the template bases which are isosteric to natural DNA bases but which lack the ability to engage in Watson-Crick (W-C) hydrogen bonding. DNA synthesis by Poleta, a low-fidelity polymerase able to replicate through DNA lesions, however, is inhibited in the presence of such an analog, suggesting a dependence of this polymerase upon W-C hydrogen bonding. Here we examine whether human Polkappa, which differs from Poleta in having a higher fidelity and which, unlike Poleta, is inhibited at inserting nucleotides opposite DNA lesions, shows less of a dependence upon W-C hydrogen bonding than does Poleta. We find that an isosteric thymidine analog is replicated with low efficiency by Polkappa, whereas a nucleobase analog lacking minor-groove H bonding potential is replicated with high efficiency. These observations suggest that both Poleta and Polkappa rely on W-C hydrogen bonding for localizing the nascent base pair in the active site for the polymerization reaction to occur, thus overcoming these enzymes' low geometric selectivity.

    View details for DOI 10.1128/MCB.25.16.7137-7143.2005

    View details for Web of Science ID 000231000800024

    View details for PubMedID 16055723

  • Chemoselective covalent coupling of oligonucleotide probes to self-assembled monolayers JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Devaraj, N. K., Miller, G. P., Ebina, W., Kakaradov, B., Collman, J. P., Kool, E. T., Chidsey, C. E. 2005; 127 (24): 8600-8601


    A chemoselective route to routinely and rapidly attach oligonucleotide probes to well-defined surfaces is presented. Cu(I) tris(benzyltriazolylmethyl)amine-catalyzed coupling of terminal acetylenes to azides on a self-assembled monolayer is used instead of traditional nucleophilic-electrophilic coupling reactions. The reaction proceeds well even in the presence of purposely introduced nucleophilic and electrophilic impurities. The density of oligonucleotide probes can be controlled by controlling the amount of azide functionality. Although most of our work was done on gold surfaces, this technique should be readily applicable to any surface on which an azide-containing monolayer can be assembled as we have preliminarily demonstrated by derivatizing azidotrimethoxysilane-modified glass slides with fluorescein-containing oligonucleotides.

    View details for DOI 10.1021/ja0514621

    View details for Web of Science ID 000229981200010

    View details for PubMedID 15954758

  • Fluorous base-pairing effects in a DNA polymerase active site CHEMISTRY-A EUROPEAN JOURNAL LAI, J. S., Kool, E. T. 2005; 11 (10): 2966-2971


    We describe selective "fluorous" effects in the active site of a DNA polymerase, by using nucleotide analogues whose pairing edges are perfluorinated. The 5'-triphosphate deoxynucleotide derivatives of DNA base analogues 2,3,4,5-tetrafluorobenzene ((F)B) and 4,5,6,7-tetrafluoroindole ((F)I), as well as hydrocarbon controls benzene (B) and indole (I), were synthesized and studied as substrates for the DNA Polymerase I Klenow fragment (KF exo-). Modified nucleotides were present in the DNA template or were supplied as nucleoside triphosphates in studies of the steady-state kinetics of single nucleotide insertion. When supplied opposite the non-natural bases in the template strand, the hydrophobic nucleoside triphosphates were incorporated by up to two orders of magnitude more efficiently than the natural deoxynucleoside triphosphates. The purine-like fluorinated indole nucleotide ((F)I) was the most efficiently inserted of the four hydrophobic analogues, with the most effective incorporation occurring opposite the pyrimidine-like tetrafluorobenzene ((F)B). In all cases, the polyfluorinated base pairs were more efficiently processed than the analogous hydrocarbon pairs. A preliminary test of polymerase extension beyond these pairs showed that only the (F)B base is appreciably extended; the inefficient extension is consistent with recently published data regarding other nonpolar base pairs. These results suggest the importance of hydrophobicity, stacking, and steric interactions in the polymerase-mediated replication of DNA base pairs that lack hydrogen bonds. These findings further suggest that the enhanced hydrophobicity of polyfluoroaromatic bases could be employed in the design of new, selective base pairs that are orthogonal to the natural Watson-Crick pairs used in replication.

    View details for DOI 10.1002/chem.20041151

    View details for Web of Science ID 000229097700008

    View details for PubMedID 15744767

  • Oligomeric fluorescent labels for DNA BIOCONJUGATE CHEMISTRY Cuppoletti, A., Cho, Y. J., Park, J. S., Strassler, C., Kool, E. T. 2005; 16 (3): 528-534


    In an effort to find fluorescent labels that have large Stokes shifts and increased emission intensity, a strategy for fluorescence labeling of DNA was explored in which multiple individual fluorophores are incorporated at adjacent positions at the end of a DNA probe. To encourage close interactions, hydrocarbon and heterocycle fluorophores were substituted at C-1 of deoxyribose, replacing the DNA base. The C-glycosides studied contained the well-known fluorophores terphenyl, pyrene, and terthiophene. For comparison, a commercial fluorescein-dU nucleotide was examined. Oligomeric labels containing up to five fluorophores were tested. Interestingly, all four dyes behaved differently on multiple substitution. Fluorescein displayed strong self-quenching properties, with the quantum yield dropping severalfold with each additional substitution and with a constant, small Stokes shift. In contrast, pyrene showed increases in quantum yield on addition of more than one fluorophore and yielded efficient long-wavelength emission on multiple substitution, with Stokes shifts of >130 nm. Oligomeric terphenyl labels gave a small progressive red shift in absorption and a marked red shift in emission wavelength and showed a strong increase in brightness with more monomers. Finally, terthiophene oligomers showed self-quenching combined with increasing Stokes shifts. Overall, the results suggest that some oligomeric fluorescent labels exhibit properties not available in common fluorescein class (or other commercial) labels, such as large Stokes shifts and increasing brightness with increasing substitution.

    View details for DOI 10.1021/bc0497766

    View details for Web of Science ID 000229274800008

    View details for PubMedID 15898718

  • Quenched probes for highly specific detection of cellular RNAs TRENDS IN BIOTECHNOLOGY Silverman, A. P., Kool, E. T. 2005; 23 (5): 225-230


    Nucleic acid-based RNA detection is a promising field in molecular biotechnology that is leading to the rapid and accurate identification of microorganisms, diagnosis of infections and imaging of gene expression. The specificity of short synthetic DNA probes raises the hope of distinguishing small differences in sequence, ultimately achieving single nucleotide resolution. Recent work using quenched fluorescently labeled oligonucleotide probes as sensors for RNA in bacterial and human cells has overcome several difficult hurdles on the way to these goals, including delivery of probes to live cells, accessing RNA sites containing a high degree of secondary structure, and eliminating many sources of background. Two new classes of quenched oligonucleotide probes, molecular beacons and quenched auto-ligation probes, have shown the most promise for in situ RNA detection. High-specificity detection, at the single-nucleotide resolution level, is now possible in solution with these classes of probes. However, for applications in intact cells, signal and background issues still need to be addressed before the full potential of these methods is achieved.

    View details for DOI 10.1016/j.tibtech.2005.03.007

    View details for Web of Science ID 000229466000004

    View details for PubMedID 15865999

  • A series of nonpolar thymidine analogues of increasing size: DNA base pairing and stacking properties JOURNAL OF ORGANIC CHEMISTRY Kim, T. W., Kool, E. T. 2005; 70 (6): 2048-2053


    [reaction: see text] We describe the properties in DNA of a set of five nonpolar nucleoside mimics in which shape is similar but size is increased gradually. The compounds vary in the size of their exocyclic substituents, which range from hydrogen to iodine, and are designed to test the steric effects of nucleosides, nucleotides, and DNA in biological systems in a systematic way. We describe the conversion of toluene, 2,4-difluorotoluene, 2,4-dichlorotoluene, 2,4-dibromotoluene, and 2,4-diiodotoluene deoxyribosides into suitably protected phosphoramidite derivatives and their incorporation into synthetic DNAs. Studies of their behavior in the context of hexamer and dodecamer duplexes were carried out, with comparison to natural thymine. Thermal melting data with compounds in 5' dangling positions showed that all five compounds stack more strongly than thymine, and all the dihalo-substituted cases stack more strongly than the unsubstituted toluene case. Stacking correlated with surface area and hydrophobicity, both of which increase across the series. In base-pairing studies, all five compounds showed destabilized pairing opposite natural bases (relative to thymine-adenine pairing), as expected. Notably, pairing among the nonpolar base analogues was considerably more stable, and some of the pairs involving the largest analogues showed stability equal to that of a natural thymine-adenine pair. The results establish the base pairing properties of a potentially useful new series of biochemical probes for DNA-protein interactions and also identify a set of new, stable hydrophobic base pairs for designed genetic pairing systems.

    View details for DOI 10.1021/jo048061t

    View details for Web of Science ID 000227566400013

    View details for PubMedID 15760186

  • A new four-base genetic helix, yDNA, composed of widened benzopyrimidine-purine pairs JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Lee, A. H., Kool, E. T. 2005; 127 (10): 3332-3338


    We describe the properties of stable DNA-like self-assembled helices composed entirely of base pairs involving two new size-expanded pyrimidines. We term this new helix geometry "yDNA" (an abbreviation of "wide DNA"). The new pyrimidine analogues, yT and yC, are increased in size by benzo-homologation and have a geometry that is distinct from previous size-expanded pyrimidines. The yT and yC deoxyribosides were incorporated into oligodeoxynucleotides designed to form four pairs: yT-A, A-yT, yC-G, and G-yC. Helices were characterized by thermal denaturation, mixing data, and circular dichroism spectra. Results showed that highly stable double-stranded helices were formed in several sequence contexts. The data further showed that yT and yC could be segregated onto one strand and used to bind to natural strands of DNA with high sequence selectivity. The combination of yC, yT, G, and A make up a new selective, self-assembling four-base genetic pairing system that functions in many respects like natural DNA, but which is structurally distinct. The results establish that multiple variants of size-expanded DNA-like helices are feasible and suggest the possibility of a future eight-base genetic system based on the yDNA geometry. Finally, the high binding selectivity, affinity, and fluorescence of yDNA strands may yield useful applications in detection of nucleic acid sequences.

    View details for DOI 10.1021/ja0430604

    View details for Web of Science ID 000227627800036

    View details for PubMedID 15755149

  • Helix-forming properties of size-expanded DNA, an alternative four-base genetic form JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Liu, H. B., Gao, J. M., Kool, E. T. 2005; 127 (5): 1396-1402


    We describe the chemical and biophysical characterization of a new four-base genetic system, in which all base pairs are larger than the natural pairs. A recent preliminary study showed that three sequences containing size-expanded DNA (xDNA) bases could form stable cooperative complexes. However, many of the standard and essential properties that natural DNA possesses were unexplored for this new class of helical assembly. We therefore undertook a study of several properties of this new genetic complex: strand stoichiometry, preferred strand polarity (i.e., parallel vs antiparallel), mismatch selectivity, base size selectivity, ionic strength dependence, fluorescence behavior, CD spectra, and sequence generality. Results showed that several sequences formed double-stranded helical complexes, and interestingly, a pyrimidine-rich strand of xDNA bases was shown to form a triple helical complex as well. A test of strand polarity showed a preference for antiparallel orientation, as does natural DNA. Mismatch and size selectivity were generally moderate to strong, with one exception. Ionic strength dependence varied by relatively small degrees from that of natural DNA, although a triple helical complex of xDNA showed more marked dependence. Spectral characteristics (fluorescence, CD) were found to be quite different than those of natural DNA, apparently because of large differences in the electronic character of the expanded pi-systems. Finally, several sequence contexts were found to form helices in a sequence-predictable manner. Two exceptions were noted and may be explained by competition from alternative folding structures and/or strong, single-stranded stacking. The viability of xDNA as an alternative genetic system and its possible biotechnological applications are discussed.

    View details for DOI 10.1021/ja0463051

    View details for Web of Science ID 000226843900031

    View details for PubMedID 15686371

  • General method for modification of liposomes for encoded assembly on supported bilayers JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Yoshina-Ishii, C., Miller, G. P., Kraft, M. L., Kool, E. T., Boxer, S. G. 2005; 127 (5): 1356-1357


    An amphiphilic oligonucleotide species ((C18)2-DNA) is presented as a generally useful reagent to display encoded tether sequences on the surface of phospholipid assemblies. (C18)2-DNA inserts into preformed vesicles and proteoliposomes of arbitrary composition, content, and origin using a simple and gentle procedure and is a significant improvement over the previously described method particularly since it allows postmodification of any phospholipid assembly without the need for special lipids carrying reactive headgroups. DNA-modified vesicles can then be tethered, via DNA hybridization, onto a supported phospholipid bilayer displaying the complementary sequence. The encoding capability of the tether can be exploited to form an array of tethered vesicles spatially defined by the DNA sequence displayed on the surface and demonstrates that (C18)2-DNA is stably associated with a membrane to allow sorting. Vesicles tethered in this way show two-dimensional mobility, reflecting the fluidity of the supporting bilayer, and promises to be a useful system with which to study vesicle-vesicle interactions.

    View details for DOI 10.1021/ja043299k

    View details for Web of Science ID 000226843900011

    View details for PubMedID 15686351

  • Size-expanded analogues of dG and dC: Synthesis and pairing properties in DNA JOURNAL OF ORGANIC CHEMISTRY Liu, H. B., Gao, J. M., Kool, E. T. 2005; 70 (2): 639-647


    We describe the completion of the set of four benzo-fused expanded DNA (xDNA) nucleoside analogues. We previously reported the development of benzo-fused analogues of dA and dT and their inclusion in an exceptionally stable new four-base genetic system, termed xDNA, in which the base pairs were expanded in size. Here we describe the preparation and properties of the second half of this nucleotide set: namely, the previously unknown dxC and dxG nucleosides. The dxC analogue was prepared from the previously reported dxT nucleoside in three steps and 57% yield. The large-sized deoxyguanosine analogue was prepared from an intermediate in the synthesis of dxA, yielding dxG in 14 steps overall (2.4%). Suitably protected versions of the deoxynucleosides were prepared for oligonucleotide synthesis following standard procedures, and they were readily incorporated into DNA by automated synthesizer. "Dangling-end" measurements revealed that the benzo-fused homologues stack considerably more strongly on neighboring DNA sequences than do their natural counterparts. Base pairing experiments with xC or xG bases showed that they pair selectively with their Watson-Crick partners, but with mild destabilization, due apparently to their larger size. Overall, the data suggest that the fluorescent xG and xC bases may be useful probes of steric effects in the study of biological nucleotide recognition mechanisms. In addition, the completion of the xDNA nucleoside set makes it possible in the future to construct full four-base xDNA strands that can target any sequence of natural DNA and RNA.

    View details for DOI 10.1021/jo048357z

    View details for Web of Science ID 000226313600034

    View details for PubMedID 15651812

  • Novel benzopyrimidines as widened analogues of DNA bases JOURNAL OF ORGANIC CHEMISTRY Lee, A. H., Kool, E. T. 2005; 70 (1): 132-140


    We report on the synthesis, stacking, and pairing properties of a new structural class of size-expanded pyrimidine nucleosides, abbreviated dyT and dyC. Their bases are benzo-homologated variants of thymine and cytosine and have a design that is distinct from a previously described class of size-expanded (xDNA) pyrimidines, with a different vector of expansion relative to the sugar. We term this new base geometry "yDNA" (a mnemonic for "wide DNA"). Both C-glycosides were prepared using Pd-mediated coupling of iodinated base derivatives with a deoxyribose precursor. As free deoxynucleosides, both dyT and dyC displayed robust fluorescence, with emission maxima at 375 and 390 nm, respectively. Both widened pyrimidines could be incorporated readily as protected phosphoramidite derivatives into synthetic oligonucleotides. Experiments in "dangling end" DNA contexts revealed that both yT and yC stack more favorably than their natural counterparts. When opposite natural bases in the context of Watson-Crick DNA were paired, the yT nucleotide formed a pair with A that was equally stable as a T-A pair, despite the mismatch in size with the neighboring natural pairs. The yC nucleotide (paired opposite G) was destabilizing by a small amount in the same context. Despite the large size of the pairs, both yT and yC were selective for their Watson-Crick complementary partners A and G, respectively. The pairing properties and fluorescence of yDNA nucleotides may lead to useful applications in the study of steric effects in DNA-protein interactions. In addition, the compounds may serve as building blocks for a large-sized artificial genetic system.

    View details for DOI 10.1021/jo0483973

    View details for Web of Science ID 000226508600016

    View details for PubMedID 15624915

  • Assembly of the complete eight-base artificial genetic helix, xDNA, and its interaction with the natural genetic system ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Gao, J. M., Liu, H. B., Kool, E. T. 2005; 44 (20): 3118-3122

    View details for DOI 10.1002/anie.200500069

    View details for Web of Science ID 000229342600026

    View details for PubMedID 15834852

  • Quenched autoligation probes allow discrimination of live bacterial species by single nucleotide differences in rRNA NUCLEIC ACIDS RESEARCH Silverman, A. P., Kool, E. T. 2005; 33 (15): 4978-4986


    Quenched autoligation (QUAL) probes are a class of self-reacting nucleic acid probes that give strong fluorescence signal in the presence of fully complementary RNAs and selectivity against single nucleotide differences in solution. Here, we describe experiments designed to test whether QUAL probes can discriminate between bacterial species by the detection of small differences in their 16S rRNA sequences. Probes were introduced into live cells using small amounts of detergent, thus eliminating the need for fixation, and fluorescence signal was monitored both by microscopy and by flow cytometry without any washing steps. The effects of probe length, modified backbone, probe concentration and growth state of the bacteria were investigated. The data demonstrate specific fluorescence discrimination between three closely related bacteria, Escherichia coli, Salmonella enterica and Pseudomonas putida, based on single nucleotide differences in their 16S rRNA. Discrimination was possible with cells in mid-log phase or in lag phase. These results suggest that QUAL probes may be useful for rapid identification of microorganisms in laboratory and clinical settings.

    View details for DOI 10.1093/nar/gki814

    View details for Web of Science ID 000231925700035

    View details for PubMedID 16284198

  • Efficient isothermal expansion of human telomeric and minisatellite repeats by Thermococcus litoralis DNA polymerase NUCLEIC ACIDS RESEARCH Hartig, J. S., Kool, E. T. 2005; 33 (15): 4922-4927


    Repeating DNA sequences, such as telomeres, centromeres, and micro- and mini-satellites, comprise 50% of the genome and play important roles in regulatory and pathogenic mechanisms. In order to study structures and functions of such repeating sequences, it is important to have simple and efficient methods for making them in vitro. Here, we describe the efficient and convenient expansion of repetitive telomeric and minisatellite DNA sequences starting from small synthetic templates to final product lengths of several hundreds to thousands of nucleotides by the thermostable DNA polymerase from Thermococcus litoralis (Vent DNA polymerase). This enzyme was so far unknown to catalyze repeat expansion. Either single-stranded or double-stranded DNAs could be produced, depending on nucleotides present. Compared to earlier results obtained with other enzymes, the expansion reaction is highly efficient both in its yield and product length, and proceeds without thermal cycling. Moreover, the products are characterized by a narrow length distribution.

    View details for DOI 10.1093/nar/gki803

    View details for Web of Science ID 000231925700030

    View details for PubMedID 16284196

  • Universal linkers for signal amplification in auto-ligating probes. Nucleic acids symposium series (2004) Abe, H., Kool, E. T. 2005: 37-38


    We reported recently oligonucleotide ligation methods for detection of DNAs and RNAs in solution and in cellular imaging. In previous systems, ligated full-length oligonucleotide products have almost native DNA structure and bind tightly with target strand, which limits the resulting signals to one per target. When small amounts of RNAs are targeted, signal amplification becomes very important issue. Here, we report on a new universal linker to destabilize ligated products in template-dependent auto-ligation, which accelerates the dissociation of ligated product from target and allows as much as 92-fold amplification of signals in DNA and RNA detection without enzymes, reagents, or thermal cycling. This signal amplification is shown in solution experiments and in solid supported assays.

    View details for PubMedID 17150621

  • Destabilizing universal linkers for signal amplification in self-ligating probes for RNA JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Abe, H., Kool, E. T. 2004; 126 (43): 13980-13986


    Recent studies have established the utility of oligonucleotide ligation methods in the detection of DNAs and RNAs in solution and in cellular imaging. Notably, the ligated full-length oligonucleotide products commonly bind to the target nucleic acid much more tightly than do the two starting half-probes, which effectively limits the resulting signals to one per target. Here, we report on a molecular strategy for destabilizing ligated products in template-promoted self-ligation reactions, thus yielding multiple signals per target. A new universal linker design is described in which a dabsyl leaving group is placed on a short alkane tether. This allows the placement of an electrophile at the end of any DNA sequence, in contrast to earlier ligation strategies, and it also speeds reaction rates by a factor of 4-5. This new class of molecular linker/activator yields as much as 92-fold amplification of signals in DNA and RNA detection, and proceeds without enzymes, added reagents, or thermal cycling. The linker is shown to destabilize the ligation product without destabilizing the transition state for ligation. This lowers product inhibition, and the target DNA or RNA thus becomes a catalyst for isothermally generating multiple signals for its detection. This enhanced signal generation is demonstrated in solution experiments and in solid supported assays.

    View details for DOI 10.1021/ja046791c

    View details for Web of Science ID 000224873600039

    View details for PubMedID 15506759

  • A set of nonpolar thymidine nucleoside analogues with gradually increasing size ORGANIC LETTERS Kim, T. W., Kool, E. T. 2004; 6 (22): 3949-3952


    [structure: see text] We describe a series of nonpolar nucleoside analogues having similar shapes and gradually increasing size. The structure of the nucleobase thymine was mimicked with toluene derivatives, replacing O2/O4 with hydrogen, fluorine, chlorine, bromine, and iodine. Glycosidic bonds were formed by reactions of lithiated 2,4-dihalotoluenes with a deoxyribonolactone derivative. Structural analysis by NMR showed similar conformations across the series. The compounds are useful for study of the biological recognition of nucleotides and nucleic acids.

    View details for DOI 10.1021/ol048487u

    View details for Web of Science ID 000224691900018

    View details for PubMedID 15496071

  • Modified DNA analogues that sense light exposure with color changes JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Gao, J. M., Watanabe, S., Kool, E. T. 2004; 126 (40): 12748-12749


    We report the discovery of a new class of light-sensing molecules. These light sensors are composed of fluorophore oligomers assembled on a DNA backbone. A combinatorial library of tetrafluorophores consisting of over 14 000 compounds was synthesized and screened for rapid responses toward light exposure. Among the most light-sensitive molecules, at least three tetramers were found to respond to light exposure with apparent color changes, rather than simple photobleaching.

    View details for DOI 10.1021/ja046910o

    View details for Web of Science ID 000224357700019

    View details for PubMedID 15469249

  • Expanded-size bases in naturally sized DNA: Evaluation of steric effects in Watson-Crick pairing JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Gao, J. M., Liu, H. B., Kool, E. T. 2004; 126 (38): 11826-11831


    We describe physicochemical properties in DNA of altered-size nucleobases that retain Watson-Crick analogous hydrogen-bonding ability. Size-expanded analogues of adenine and thymine (xA and xT, respectively, which are expanded by benzo-fusion) were incorporated into natural DNA oligonucleotides, and their effects on helix stability were measured. Base stacking studies revealed that the two stretched analogues stack much more strongly than do their naturally sized counterparts. In contrast to this, pairing studies showed that single substitutions of the new bases are destabilizing to the natural helix as compared to A or T in standard A-T pairs in the same context, unless multiple adjacent substitutions are used. Interestingly, the size-expanded bases displayed selective recognition of the hydrogen-bonding complementary partners, suggesting that Watson-Crick analogous pairs were still formed despite local backbone strain. In an attempt to compensate for the added size of the expanded adenine, we tested a formamide deoxynucleoside, which Leonard proposed as a shortened thymine analogue (F(o)). Data showed, however, that this compound adopts a conformation unfavorable for pairing. On the basis of the combined thermodynamic data, we estimate the energetic cost of the 2.4 A stretching of an isolated base pair in DNA at ca. +1 to 2 kcal/mol. Notably, during the pairing studies, the two size-expanded nucleobases were found to display significant changes in fluorescence on formation of stacked versus unstacked structures, suggesting possible applications in probing nucleic acid structures and biochemical mechanisms.

    View details for DOI 10.1021/ja048499a

    View details for Web of Science ID 000224103900034

    View details for PubMedID 15382917

  • Solution structure of xDNA: A paired genetic helix with increased diameter JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Liu, H. B., Lynch, S. R., Kool, E. T. 2004; 126 (22): 6900-6905


    We describe the structure in aqueous solution of an extended-size DNA-like duplex with base pairs that are approximately 2.4 A longer than those of DNA. Deoxy-lin-benzoadenosine (dxA) was employed as a dA analogue to form hydrogen-bonded base pairs with dT. The 10mer self-complementary extended oligodeoxynucleotide 5'-d(xATxAxATxATTxAT) forms a much more thermodynamically stable duplex than the corresponding DNA sequence, 5'-d(ATAATATTAT). NMR studies show that this extended DNA (xDNA) retains many features of natural B-form DNA, but with a few structural alterations due to its increased helical diameter. The results give insight into the structural plasticity of the natural DNA backbone and lend insight into the evolutionary origins of the natural base pairs. Finally, this structural study confirms the hypothesis that extended nucleobase analogues can form stable DNA-like structures, suggesting that alternative genetic systems might be viable for storage and transfer of genetic information.

    View details for DOI 10.1021/ja0497835

    View details for Web of Science ID 000221828200028

    View details for PubMedID 15174859

  • Hydrolysis of RNA/DNA hybrids containing non-polar pyrimidine isosteres defines regions essential for HIV-1 polypurine tract selection Rausch, J., Qu, J., Yi-Brunozzi, H. Y., Kool, E., Le Grice, S. FEDERATION AMER SOC EXP BIOL. 2004: C135-C136
  • Versatile 5 '-functionalization of oligonucleotides on solid support: Amines, azides, thiols, and thioethers via phosphorus chemistry JOURNAL OF ORGANIC CHEMISTRY Miller, G. P., Kool, E. T. 2004; 69 (7): 2404-2410


    Although the preparation of conjugates of oligonucleotides is by now commonplace, existing methods (usually utilizing thiols or primary amines) are generally expensive, and often require postsynthetic reaction with the DNA followed by a separate purification. Here we describe simple procedures for a broad set of direct 5'-end (5'-terminal carbon) functionalizations of DNA oligonucleotides while they remain on the synthesizer column. 5'-Iodinated oligonucleotides (prepared by an automated cycle as previously reported) are converted directly to 5'-azides, 5'-thiocarbamates, and alkyl and aryl 5'-thioethers in high yields. Further, we demonstrate high-yielding conversions of DNA-azides to 5'-amines, and of thiocarbamates to 5'-thiols. Finally, we report a new, one-pot conversion of naturally substituted 5'-OH oligonucleotides (again on the solid support) to 5'-amino-oligonucleotides. All of the above reactions are demonstrated in multiple sequence contexts. Most of the procedures are automatable.

    View details for Web of Science ID 000220506200025

    View details for PubMedID 15049637

  • Selective pairing of polyfluorinated DNA bases JOURNAL OF THE AMERICAN CHEMICAL SOCIETY LAI, J. S., Kool, E. T. 2004; 126 (10): 3040-3041


    Novel selective non-hydrogen-bonding DNA base pairs utilizing fluorinated nucleoside analogues have been investigated. Melting studies of DNA duplexes containing 2,3,4,5-tetrafluorobenzene and 4,5,6,7-tetrafluoroindole bases on opposite strands show greater stabilization of the duplex compared with nonfluorinated hydrocarbon controls. Overall, these hydrophobic analogues are destabilizing compared with natural base pairs but are stabilizing compared with natural base mismatches. Such selective pairing may be due to solvent avoidance of these hydrophobic structures, burying their surfaces within the duplex. Our findings suggest that polyfluoroaromatic bases might be employed as a new, selective base-pairing system orthogonal to the natural genetic system.

    View details for DOI 10.1021/ja039571s

    View details for Web of Science ID 000220192000018

    View details for PubMedID 15012120

  • Quenched auto-ligating DNAs: Multicolor identification of nucleic acids at single nucleotide resolution JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Sando, S., Abe, H., Kool, E. T. 2004; 126 (4): 1081-1087


    We describe the synthesis and study of multicolor quenched autoligating (QUAL) probes for identification and discrimination of closely related RNA and DNA sequences in solution and in bacteria. In these probes, a dabsyl quencher doubles as an activator in the oligonucleotide-joining reaction. The oligonucleotides remain dark until they bind at adjacent sites, and "light up" on nucleophilic displacement of the dabsyl probe by the phosphorothioate probe. Four fluorescent dye conjugates were prepared and tested with probes and targets that differ by one nucleotide. Experiments on polymer beads show clear color-based discrimination of DNAs added in solution. Two-color quenched probe pairs were then tested in the discrimination of 16S rRNA sequences in Escherichia coli. Single nucleotide resolution was achieved in the cells with green/red QUAL probes, allowing identification of a one-base sequencing error in the 16S rRNA database. Finally, QUAL probes were successfully applied in live bacterial cells. The method requires only incubation followed by fluorescence imaging, and requires no enzymes, added reagents, cross-linking, fixing, or washes. Because probes must bind side-by-side to generate signal, there is little or no interference from unintended protein binding, which can occur with other probe types. The results suggest that QUAL probes may be of general use in the detection and identification of sequences in solution, on microarrays, and in microorganisms.

    View details for DOI 10.1021/ja038665z

    View details for Web of Science ID 000188534200035

    View details for PubMedID 14746476

  • Toward a new genetic system with expanded dimensions: Size-expanded analogues of deoxyadenosine and thymidine JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Liu, H. B., Gao, J. M., Maynard, L., Saito, Y. D., Kool, E. T. 2004; 126 (4): 1102-1109


    We describe the design, preparation, and properties of two key building blocks of a size-expanded genetic system. Nucleoside analogues of the natural nucleosides dA and dT are reported in which the fusion of a benzo ring increases their size by ca. 2.4 A. The expanded dA analogue (dxA), having a tricyclic base, was first reported by Leonard nearly three decades ago. We describe a shortened and more efficient approach to this compound. The expanded dT analogue (dxT), a methylquinazolinedione C-glycoside, was previously unknown; we describe its preparation in eight steps from 5-methylanthranilic acid. The key glycoside bond formation employed Pd-mediated coupling of an aryl iodide precursor with a dihydrofuran derivative of deoxyribose. Both nucleosides are shown to be efficient fluorophores, emitting light in the blue-violet range. The base-protected phosphoramidite derivatives were prepared, and short oligonucleotides containing them were characterized. The two size-expanded nucleosides are key components of a new four-base genetic system designed to form helical paired structures having a diameter greater than that of natural DNA. Elements of the design of this expanded genetic molecule, termed xDNA, are discussed, including the possibility of up to eight base pairs of information storage capability.

    View details for DOI 10.1021/ja038384r

    View details for Web of Science ID 000188534200038

    View details for PubMedID 14746479

  • Direct comparison of A- and T-strand minor groove interactions in DNA curvature at a tracts BIOCHEMISTRY Maki, A. S., Kim, T. W., Kool, E. T. 2004; 43 (4): 1102-1110


    To investigate the relative contributions of minor-groove electrostatic interactions in the mechanism of A-tract DNA curvature, we carried out experiments with modified DNA bases in both strands of the tract. We employed 3-deazaadenine nucleoside (D), which lacks the adenine N3 nitrogen in the minor groove and thus cannot act as an electron donor, as well as difluorotoluene (F), a nonpolar thymine mimic. The effects of these analogues in A-tract curvature were quantified using ligation ladder gel mobility methods developed by Crothers and by Maher. Through single substitutions of D in A(5) tracts, we found that this analogue results in decreased curvature only when situated toward the 3' end of the tract. This is distinct from the behavior in the T-rich strand where F substitution causes the greatest reductions in curvature toward the 5' end. To test for cooperative pairwise effects, we also studied 10 different D + F double substitutions and found evidence supporting a number of localized cooperative electrostatic interactions but not between the two most sensitive sites in the opposite strands. These results suggest that there are two discrete locations in the A-tract minor groove where electrostatic interactions are important in causing curvature: one near the 5' end of the T-rich strand, and one near the 3' end of the A-rich strand. The results are consistent with an important role of localized cations in the minor groove. Possible effects of groove solvation and stacking at the A-tract junction are also discussed.

    View details for DOI 10.1021/bi035340m

    View details for Web of Science ID 000188504800032

    View details for PubMedID 14744156

  • Solid-phase synthesis and screening of macrocyclic nucleotide-hybrid compounds targeted to hepatitis CNS5B CHEMISTRY-A EUROPEAN JOURNAL Smietana, M., Johnson, R. B., Wang, Q. M., Kool, E. T. 2004; 10 (1): 173-181


    A convergent strategy for the synthesis of cyclic nucleotide-hybrid molecules on controlled pore glass is reported. A major advantage of the approach is the lack of restrictions on the sequence and structural variation, allowing the incorporation of modified ribonucleosides (such as 2'-OMe-ribonucleotides), as well as threoninol derivatives. This methodology allows a fully automated assembly by means of standard phosphoramidite chemistry and is based on a recently published procedure for the preparation of cyclic oligodinucleotides in the DNA series (M. Smietana, E. T. Kool, Angew. Chem. 2002, 114, 3856-3859; Angew. Chem. Int. Ed. Engl. 2002, 41, 3704-3707). A library of potential cyclic hybrid inhibitor compounds targeting hepatitis C virus NS5B enzyme (the replicating polymerase of HCV) was generated by means of the parallel-pool strategy. Screening of the library revealed that cyclic hybrid c(C(OME)EthenodA) was a significant inhibitor of NS5B, with an IC(50) of 40 microM. Preliminary structure-activity studies of this lead compound are described.

    View details for DOI 10.1002/chem.200305402

    View details for Web of Science ID 000188087800017

    View details for PubMedID 14695562

  • yDNA: A new geometry for size-expanded base pairs ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Lu, H. G., He, K. Z., Kool, E. T. 2004; 43 (43): 5834-5836

    View details for DOI 10.1002/anie.200461036

    View details for Web of Science ID 000225204100024

    View details for PubMedID 15523708

  • Small circular DNAs for synthesis of the human telomere repeat: varied sizes, structures and telomere-encoding activities NUCLEIC ACIDS RESEARCH Hartig, J. S., Kool, E. T. 2004; 32 (19)


    We describe the construction, structural properties and enzymatic substrate abilities of a series of circular DNA oligonucleotides that are entirely composed of the C-rich human telomere repeat, (CCCTAA)n. The nanometer-sized circles range in length from 36 to 60 nt, and act as templates for synthesis of human telomere repeats in vitro. The circles were constructed successfully by the application of a recently developed adenine-protection strategy, which allows for cyclization/ligation with T4 DNA ligase. Thermal denaturation studies showed that at pH 5.0, all five circles form folded structures with similar stability, while at pH 7.0 no melting transitions were seen. Circular dichroism spectra at the two pH conditions showed evidence for i-motif structures at the lower pH value. The series was tested as rolling circle templates for a number of DNA polymerases at pH = 7.3-8.5, using 18mer telomeric primers. Results showed that surprisingly small circles were active, although the optimum size varied from enzyme to enzyme. Telomeric repeats >1000 nt in length could be synthesized in 1 h by the Klenow (exo-) DNA polymerase. The results establish a convenient way to make long human telomeric repeats for in vitro study of their folding and interactions, and establish optimum molecules for carrying this out.

    View details for DOI 10.1093/nar/gnh149

    View details for Web of Science ID 000225259100011

    View details for PubMedID 15520461

  • Probing the requirements for recognition and catalysis in fpg and MutY with nonpolar adenine isosteres JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Francis, A. W., Helquist, S. A., Kool, E. T., David, S. S. 2003; 125 (52): 16235-16242


    The Escherichia coli DNA repair enzymes Fpg and MutY are involved in the prevention of mutations resulting from 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG) in DNA. The nonpolar isosteres of 2'-deoxyadenosine, 4-methylbenzimidazole beta-deoxynucleoside (B), and 9-methyl-1H-imidazo[4,5-b]pyridine beta-deoxynucleoside (Q), were used to examine the importance of hydrogen bonding within the context of DNA repair. Specifically, the rate of base removal under single-turnover conditions by the MutY and Fpg glycosylases from duplexes containing OG:B and OG:Q mismatches, relative to OG:A mismatches, was evalulated. The reaction of Fpg revealed a 5- and 10-fold increase in rate of removal of OG from duplexes containing OG:B and OG:Q base pairs, respectively, relative to an OG:A mispair. These results suggest that the lack of the ability to hydrogen bond to the opposite base facilitates removal of OG. In contrast, adenine removal catalyzed by MutY was much more efficient from an OG:A mispair-containing duplex (k2 = 12 +/- 2 min(-1)) compared to the removal of B from an OG:B duplex (k(obs) < 0.002 min(-1)). Surprisingly, MutY was able to catalyze base removal from the OG:Q-containing substrate (k2 = 1.2 +/- 0.2 min(-1)). Importantly, the B and Q analogues are not deleterious to high-affinity DNA binding by MutY. In addition, the B and Q analogues are more susceptible to acid-catalyzed depurination illustrating that the enzyme-catalyzed mechanism is distinct from the nonenzymatic mechanism. Taken together, these results point to the importance of both N7 and N3 in the mechanism of adenine excision catalyzed by MutY.

    View details for DOI 10.1021/ja0374426

    View details for Web of Science ID 000187574800031

    View details for PubMedID 14692765

  • 5'-Iodination of solid-phase-linked oligodeoxyribonucleotides. Current protocols in nucleic acid chemistry / edited by Serge L. Beaucage ... [et al.] Kool, E. T., Miller, G. P. 2003; Chapter 4: Unit 4 19-?


    5'-Iodinated oligodeoxyribonucleotides readily react with 3'-phosphorothioated DNA in the presence of a complementary template to yield a conjugate that is identical to natural DNA in every respect except that one oxygen atom in the phosphodiester backbone is replaced by a sulfur atom. The 5'-iodo group is easily converted to a variety of other functional groups and will quickly react with thiol-containing labels to yield stable thioether conjugates. This unit presents manual and automated procedures for converting the 5'-hydroxyl of protected CPG-bound oligodeoxyribonucleotides to an iodo group and for releasing and purifying the products.

    View details for DOI 10.1002/0471142700.nc0419s14

    View details for PubMedID 18428909

  • A four-base paired genetic helix with expanded size SCIENCE Liu, H. B., Gao, J. M., Lynch, S. R., Saito, Y. D., Maynard, L., Kool, E. T. 2003; 302 (5646): 868-871


    We describe a new molecular class of genetic-pairing system that has a native DNA backbone but has all four base pairs replaced by new, larger pairs. The base pairs include size-expanded analogs of thymine and of adenine, both extended by the width of a benzene ring (2.4 A). The expanded-diameter double helices are more thermodynamically stable than the Watson-Crick helix, likely because of enhanced base stacking. Structural data confirm a right-handed, double-stranded, and base-paired helical form. Because of the larger base size, all the pairs of this helical system are fluorescent, which suggests practical applications in detection of natural DNA and RNA. Our findings establish that there is no apparent structural or thermodynamic prohibition against genetic systems having sizes different from the natural one.

    View details for Web of Science ID 000186258000050

    View details for PubMedID 14593180

  • Hydrolysis of RNA/DNA hybrids containing nonpolar pyrimidine isosteres defines regions essential for HIV type 1 polypurine tract selection PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Rausch, J. W., Qu, J., Yi-Brunozzi, H. Y., Kool, E. T., Le Grice, S. F. 2003; 100 (20): 11279-11284


    Both x-ray crystallography and chemical footprinting indicate that bases of the HIV type 1 (HIV-1) polypurine tract (PPT)-containing RNA/DNA hybrid deviate from standard Watson-Crick base pairing. However, the contribution of these structural anomalies to the accuracy of plus-strand primer selection by HIV-1 reverse transcriptase is not immediately clear. To address this issue, DNA templates harboring single and pairwise non-hydrogen-bonding isosteres of cytosine (2-fluoro-4-methylbenzene deoxyribonucleoside) and thymine (2,4-difluoro-5-methylbenzene deoxyribonucleoside) were synthesized and hybridized to PPT-containing RNA primers as a means of locally removing hydrogen bonding and destabilizing paired structure. Cleavage of these hybrids was examined with p66/p51 HIV-1 reverse transcriptase and a mutant carrying an alteration in the p66 RNase H primer shown to specifically impair PPT processing. Analog insertion within the PPT (rG):(dC) and central (rA):(dT) tracts repositioned the RNase H domain such that the RNA/DNA hybrid was cleaved 3-4 bp from the site of insertion, a distance corresponding closely to the spatial separation between the catalytic center and RNase H primer grip. However, PPT processing was significantly impaired when the junction between these tracts was substituted. Substitutions within the upstream (rA):(dT) tract, where maximum distortion had previously been observed, destroyed PPT processing. Collectively, our scanning mutagenesis approach implicates multiple regions of the PPT in the accuracy with which it is excised from (+) U3 RNA and DNA, and also provides evidence for close cooperation between the RNase H primer grip and catalytic center in achieving this cleavage.

    View details for DOI 10.1073/pnas.1932546100

    View details for Web of Science ID 000185685700021

    View details for PubMedID 12972638

  • Probing electrostatic factors in DNA stacking with fluorinated aromatic C-deoxyribonucleosides. Lai, J. S., Qu, J., Kool, E. T. AMER CHEMICAL SOC. 2003: U233-U233
  • Requirement of Watson-Crick hydrogen bonding for DNA synthesis by yeast DNA polymerase eta MOLECULAR AND CELLULAR BIOLOGY Washington, M. T., Helquist, S. A., Kool, E. T., Prakash, L., Prakash, S. 2003; 23 (14): 5107-5112


    Classical high-fidelity DNA polymerases discriminate between the correct and incorrect nucleotides by using geometric constraints imposed by the tight fit of the active site with the incipient base pair. Consequently, Watson-Crick (W-C) hydrogen bonding between the bases is not required for the efficiency and accuracy of DNA synthesis by these polymerases. DNA polymerase eta (Poleta) is a low-fidelity enzyme able to replicate through DNA lesions. Using difluorotoluene, a nonpolar isosteric analog of thymine unable to form W-C hydrogen bonds with adenine, we found that the efficiency and accuracy of nucleotide incorporation by Poleta are severely impaired. From these observations, we suggest that W-C hydrogen bonding is required for DNA synthesis by Poleta; in this regard, Poleta differs strikingly from classical high-fidelity DNA polymerases.

    View details for DOI 10.1128/MCB.23.14.5107-5112.2003

    View details for Web of Science ID 000183958100032

    View details for PubMedID 12832493

  • High-fidelity in vivo replication of DNA base shape mimics without Watson-Crick hydrogen bonds PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Delaney, J. C., Henderson, P. T., Helquist, S. A., Morales, J. C., Essigmann, J. M., Kool, E. T. 2003; 100 (8): 4469-4473


    We report studies testing the importance of Watson-Crick hydrogen bonding, base-pair geometry, and steric effects during DNA replication in living bacterial cells. Nonpolar DNA base shape mimics of thymine and adenine (abbreviated F and Q, respectively) were introduced into Escherichia coli by insertion into a phage genome followed by transfection of the vector into bacteria. Genetic assays showed that these two base mimics were bypassed with moderate to high efficiency in the cells and with very high efficiency under damage-response (SOS induction) conditions. Under both sets of conditions, the T-shape mimic (F) encoded genetic information in the bacteria as if it were thymine, directing incorporation of adenine opposite it with high fidelity. Similarly, the A mimic (Q) directed incorporation of thymine opposite itself with high fidelity. The data establish that Watson-Crick hydrogen bonding is not necessary for high-fidelity replication of a base pair in vivo. The results suggest that recognition of DNA base shape alone serves as the most powerful determinant of fidelity during transfer of genetic information in a living organism.

    View details for DOI 10.1073/pnas.0837277100

    View details for Web of Science ID 000182306100026

    View details for PubMedID 12676985

  • DNA curvature at A tracts containing a non-polar thymine mimic NUCLEIC ACIDS RESEARCH Maki, A., Brownewell, F. E., Liu, D., Kool, E. T. 2003; 31 (3): 1059-1066


    We report the first experimental probing of electrostatic interactions on the pyrimidine side of a bent A tract. Although the curvature of short A tracts (A4-A6) has long been studied, its physical origins remain under debate. Current hypotheses include the influence of major-groove hydrogen bonds between propeller-twisted base pairs, electrostatic effects of closely associated minor-groove cations, effects of minor-groove solvation, and stacking effects at the junctions adjacent to the A tract. We investigated this problem through the substitution of thymidines in A5 tracts by difluorotoluene deoxynucleoside (F), a non-polar molecule of the same size and shape which lacks hydrogen bonding and metal-ion complexing capabilities. Ligation experiments with phased A tracts demonstrated that F substitution has asymmetric effects on the bend angle. The strongest effects occurred at the second and third thymines where curvature was reduced from 19.8 degrees to 5.3 degrees and 9.6 degrees, respectively. Moderate effects were observed with substitutions at positions 1 and 4, while substitution at position 5 had no effect on bend angle. The results support the hypothesis that highly localized electrostatic interactions are a principal cause of A-tract curvature. Furthermore, they are most consistent with the notion that local metal-ion complexation at O2 of thymine is a strong component of these interactions.

    View details for DOI 10.1093/nar/gkg172

    View details for Web of Science ID 000181114500036

    View details for PubMedID 12560504

  • Kinetics and binding of the thymine-DNA Mig-Mth, with mismatch-containing mismatch glycosylase, DNA substrates DNA REPAIR Begley, T. J., Haas, B. J., Morales, J. C., Kool, E. T., Cunningham, R. P. 2003; 2 (1): 107-120
  • Fluorinated DNA bases as probes of electrostatic effects in DNA base stacking ANGEWANDTE CHEMIE-INTERNATIONAL EDITION LAI, J. S., Qu, J., Kool, E. T. 2003; 42 (48): 5973-5977

    View details for DOI 10.1002/anie.200352531

    View details for Web of Science ID 000187570400011

    View details for PubMedID 14679546

  • A porphyrin C-nucleoside incorporated into DNA ORGANIC LETTERS Morales-Rojas, H., Kool, E. T. 2002; 4 (25): 4377-4380


    [reaction: see text] A free porphyrin coupled on 2-deoxy-D-ribose was synthesized and incorporated into DNA via phosphoramidite chemistry. Substitution at the ends of a 5'-modified self-complementary duplex was found to be thermally and thermodynamically stabilizing. The porphyrin moiety strongly intercalates in the duplex when located near the center, and retains its fluorescence properties in DNA.

    View details for DOI 10.1021/ol0267376

    View details for Web of Science ID 000179715900001

    View details for PubMedID 12465891

  • Artificial human telomeres from DNA nanocircle templates PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Lindstrom, U. M., Chandrasekaran, R. A., Orbai, L., Helquist, S. A., Miller, G. P., Oroudjev, E., HANSMA, H. G., Kool, E. T. 2002; 99 (25): 15953-15958


    Human telomerase is a reverse-transcriptase enzyme that synthesizes the multikilobase repeating hexamer telomere sequence (TTAGGG)n at the ends of chromosomes. Here we describe a designed approach to mimicry of telomerase, in which synthetic DNA nanocircles act as essentially infinite catalytic templates for efficient synthesis of long telomeres by DNA polymerase enzymes. Results show that the combination of a nanocircle and a DNA polymerase gives a positive telomere-repeat amplification protocol assay result for telomerase activity, and similar to the natural enzyme, it is inhibited by a known telomerase inhibitor. We show that artificial telomeres can be engineered on human chromosomes by this approach. This strategy allows for the preparation of synthetic telomeres for biological and structural study of telomeres and proteins that interact with them, and it raises the possibility of telomere engineering in cells without expression of telomerase itself. Finally, the results provide direct physical support for a recently proposed rolling-circle mechanism for telomerase-independent telomere elongation.

    View details for DOI 10.1073/pnas.252396199

    View details for Web of Science ID 000179783400026

    View details for PubMedID 12444252

  • Replacing the nucleohases in DNA with designer molecules ACCOUNTS OF CHEMICAL RESEARCH Kool, E. T. 2002; 35 (11): 936-943


    DNA is not only a carrier of genetic information, but it is also a versatile supramolecular scaffold, arranging smaller organic structures into predesigned geometries. Herein are discussed molecular strategies in which the natural DNA bases on the sugar-phosphate backbone are replaced by other molecules. Some of the base replacements under study include fluorophores, ligands for metals, helix stabilizers, and DNA base shape mimics.

    View details for Web of Science ID 000179410200003

    View details for PubMedID 12437318

  • A simple method for electrophilic functionalization of DNA ORGANIC LETTERS Miller, G. P., Kool, E. T. 2002; 4 (21): 3599-3601


    [reaction: see text] An extremely simple and versatile method for placing an electrophilic functional group (iodide) at the 5' end of oligodeoxyribonucleotides is described. The reaction is carried out while the protected oligodeoxyribonucleotide remains on a solid support and utilizes inexpensive iodination chemistry. We demonstrate that this reaction can be automated on a DNA synthesizer as the last step of DNA synthesis.

    View details for DOI 10.1021/ol0264915

    View details for Web of Science ID 000178522700013

    View details for PubMedID 12375897

  • Libraries of composite polyfluors built from fluorescent deoxyribosides JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Gao, J. M., Strassler, C., Tahmassebi, D., Kool, E. T. 2002; 124 (39): 11590-11591


    We report on a new class of water-soluble fluorescent molecules (polyfluors) that are composed of multiple individual fluorophores assembled on a DNA-like backbone. Four fluorophore deoxyribosides were synthesized, and these individual molecules were assembled into oligofluor strings on a DNA synthesizer. A library of 256 tetrafluors was generated by split and pool methods on polystyrene beads. Images of the library under a fluorescence microscope revealed at least 40-50 different hues and intensities. Selected tetrafluors were resynthesized in pure form in solution and displayed properties, such as large Stokes shifts, that individual fluorophores do not have.

    View details for DOI 10.1021/ja027197a

    View details for Web of Science ID 000178317100015

    View details for PubMedID 12296712

  • An orthogonal oligonucleotide protecting group strategy that enables assembly of repetitive or highly structured DNAs NUCLEIC ACIDS RESEARCH Lindstrom, U. M., Kool, E. T. 2002; 30 (19)


    A general problem that exists in the assembly of large and organized DNA structures from smaller fragments is secondary structure that blocks or prevents it. For example, it is common to assemble longer synthetic DNA and RNA fragments by ligation of smaller synthesized units, but blocking secondary structure can prevent the formation of the intended complex before enzymatic ligation can occur. In addition, there is a general need for protecting groups that would block reactivity of some DNA bases in a sequence, leaving others free to react or hybridize. Here we describe such a strategy. The approach involves the protecting group dimethylacetamidine (Dma), which we show to remain intact on exocyclic amines of adenine bases while other bases carrying commercially available 'ultra mild deprotection' protecting groups are removed by potassium carbonate in methanol. The intact Dma groups prevent unwanted hybridization at undesired sites, thus encouraging it to occur where intended, and allowing for successful ligations. The Dma group is then deprotected by treatment with ammonia in methanol. Other common amine protecting groups such as benzoyl and allyloxycarbonyl were not successful in such a strategy, at least in part because they did not prevent hybridization. We demonstrate the method in the synthesis of a circular 54mer oligonucleotide composed of nine human telomere repeats, which was not possible to assemble by conventional methods.

    View details for Web of Science ID 000178558500006

    View details for PubMedID 12364618

  • A highly effective nonpolar isostere of deoxyguanosine: Synthesis, structure, stacking, and base pairing JOURNAL OF ORGANIC CHEMISTRY O'Neill, B. M., Ratto, J. E., Good, K. L., Tahmassebi, D. C., Helquist, S. A., Morales, J. C., Kool, E. T. 2002; 67 (17): 5869-5875


    We describe the preparation and structure of the deoxyribonucleoside of 4-fluoro-6-methylbenzimidazole, abbreviated dH (8), which acts as a close shape mimic of the nucleoside deoxyguanosine. The nucleoside is prepared from 2-fluoro-4-methylaniline in seven steps. The X-ray crystal structure reveals a (-sc) glycosidic orientation, an S conformation for the deoxyribose moiety, and quite close shape mimicry of guanine by the substituted benzimidazole. Conformational studies by (1)H NMR and (1)H-(1)H ROESY experiments reveal an S-type conformation and an anti glycosidic orientation in solution (D(2)O), essentially the same as that of deoxyguanosine. Base-stacking studies in a "dangling end" context reveal that the benzimidazole base mimic stacks more strongly than all four natural bases, and more strongly than its counterpart guanine by 1.1 kcal/mol. Base-pairing studies in a 12mer DNA duplex show that, like other nonpolar nucleoside isosteres, H is destabilizing and nonselective when paired opposite natural bases. However, when paired opposite another nonpolar isostere, difluorotoluene (F), a mimic of thymine, the pair exhibits stability approaching that of its natural analogue, a G-T (wobble) base pair. The nucleoside analogue dH will be useful in studies of protein-DNA interactions, and the H-F base pair will serve as a structurally and thermodynamically close mimic of G-T in studies of DNA mismatch repair enzymes.

    View details for DOI 10.1021/jo025884e

    View details for Web of Science ID 000177559800002

    View details for PubMedID 12182615

  • Imaging of RNA in bacteria with self-ligating quenched probes JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Sando, S., Kool, E. T. 2002; 124 (33): 9686-9687


    We report on the application of a new class of oligonucleotide reporter probes, QUAL probes, that "light up" when a nucleophilic phosphorothioate probe binds adjacent to a dabsyl-quenched probe. These self-ligating DNA probes were used for sequence-specific detection of 16S rRNA in Escherichia coli cells. Strong fluorescence was observed only when the phosphorothioate and quenched dabsyl probes bind side-by-side on a 16S rRNA target. The results demonstrate the use of QUAL probes to detect specific RNA sequences in bacterial cells without enzymes and without washing steps.

    View details for DOI 10.1021/ja026649g

    View details for Web of Science ID 000177455000015

    View details for PubMedID 12175209

  • DNA binding properties of the yeast Msh2-Msh6 and Mlh1-Pms1 heterodimers BIOLOGICAL CHEMISTRY Drotschmann, K., Hall, M. C., Shcherbakova, P. V., Wang, H., Erie, D. A., Brownewell, F. R., Kool, E. T., Kunkel, T. A. 2002; 383 (6): 969-975


    We describe here our recent studies of the DNA binding properties of Msh2-Msh6 and Mlh1-Pms1, two protein complexes required to repair mismatches generated during DNA replication. Mismatched DNA binding by Msh2-Msh6 was probed by mutagenesis based on the crystal structure of the homologous bacterial MutS homodimer bound to DNA. The results suggest that several amino acid side chains inferred to interact with the DNA backbone near the mismatch are critical for repair activity. These contacts, which are different in Msh2 and Msh6, likely facilitate stacking and hydrogen bonding interactions between side chains in Msh6 and the mismatched base, thus stabilizing a kinked DNA conformation that permits subsequent repair steps coordinated by the Mlh1-Pms1 heterodimer. Mlh1-Pms1 also binds to DNA, but independently of a mismatch. Mlh1-Pms1 binds short DNA substrates with low affinity and with a slight preference for single-stranded DNA. It also binds longer duplex DNA molecules, but with a higher affinity indicative of cooperative binding. Indeed, imaging by atomic force microscopy reveals cooperative DNA binding and simultaneous interaction with two DNA duplexes. The novel DNA binding properties of Mlh1-Pms1 may be relevant to signal transduction during DNA mismatch repair and to recombination, meiosis and cellular responses to DNA damage.

    View details for Web of Science ID 000176571900010

    View details for PubMedID 12222686

  • Quencher as leaving group: Efficient detection of DNA-joining reactions JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Sando, S., Kool, E. T. 2002; 124 (10): 2096-2097


    We describe a new fluorescence reporting strategy in which dabsyl, a well-known quencher, activates a hydroxyl group in a probe to convert it to a leaving group. When a nucleophilic phosphorothioate probe binds adjacent to a dabsyl quenched probe, autoligation occurs, releasing the quencher, and lighting up the probes, This signal change can be used to detect single nucleotide differences in DNA without enzymes or reagents.

    View details for DOI 10.1021/ja017328s

    View details for Web of Science ID 000174469600012

    View details for PubMedID 11878946

  • Efficient bacterial transcription of DNA nanocircle vectors with optimized single-stranded promoters PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ohmichi, T., Maki, A., Kool, E. T. 2002; 99 (1): 54-59


    We describe experiments aimed at establishing whether circular single-stranded DNAs can form promoters for bacterial transcription from small folded motifs. In vitro selection experiments were carried out on circular 103-nt DNA libraries encoding 40-nt randomized sequences as well as self-processing hammerhead ribozymes. Rounds of rolling circle transcription, reverse transcription-PCR, and recyclization were carried out to optimize transcription efficiency. Sequences were identified that are 80-fold more actively transcribed than the initial library by E. coli RNA polymerase (RNAP). The selected motifs were found to be more active than canonical E. coli promoters in the same context. Experiments also demonstrated that a single-stranded pseudopromoter identified by this selection can be transplanted to other circular DNA contexts and retain transcriptional activity. Results suggest that the promoter is localized in a short ( approximately 40 nt) hairpin, which is smaller than canonical E. coli promoters. To test whether this pseudopromoter was active in bacterial cells, a synthetic DNA nanocircle vector encoding a ribozyme targeted to a site in the marA drug resistance gene was constructed to contain an optimized single-stranded promoter. It is shown that this DNA circle can act as a "Trojan horse" in E. coli, being actively transcribed by the cellular RNAP and producing ribozymes that cleave a sequence in the marA drug resistance gene. The use of optimized single-stranded promoters in combination with synthetic nanocircle DNA vectors represents a potentially useful way to engender the synthesis of biologically active RNAs in living cells.

    View details for Web of Science ID 000173233300013

    View details for PubMedID 11752404

  • Active site tightness and substrate fit in DNA replication ANNUAL REVIEW OF BIOCHEMISTRY Kool, E. T. 2002; 71: 191-219


    Various physicochemical factors influence DNA replication fidelity. Since it is now known that Watson-Crick hydrogen bonds are not necessary for efficient and selective replication of a base pair by DNA polymerase enzymes, a number of alternative physical factors have been examined to explain the efficiency of these enzymes. Among these factors are minor groove hydrogen bonding, base stacking, solvation, and steric effects. We discuss the concept of active site tightness in DNA polymerases, and consider how it might influence steric (size and shape) effects of nucleotide selection in synthesis of a base pair. A high level of active site tightness is expected to lead to higher fidelity relative to proteins with looser active sites. We review the current data on what parts and dimensions of active sites are most affected by size and shape, based on data with modified nucleotides that have been examined as polymerase substrates. We also discuss recent data on nucleotide analogs displaying higher fidelity than the natural ones. The published data are discussed with a view toward testing this sterically based hypothesis and unifying existing observations into a narrowly defined range of effects.

    View details for DOI 10.1146/annurev.biochem.71.110601.135453

    View details for Web of Science ID 000177352600009

    View details for PubMedID 12045095

  • Nonenzymatic DNA ligation in Escherichia coli cells. Nucleic acids research. Supplement (2001) Sando, S., Kool, E. T. 2002: 121-122


    We report on a new fluorescence reporting strategy in which dabsyl, a well-known quencher, activates a hydroxyl group in a probe to convert it to a leaving group. When a nucleophilic phosphorothioate probe binds adjacent to a dabsyl quenched probe, autoligation occurs, releasing the quencher, and lighting up the probes. These self-ligating DNA probes were used for sequence-specific detection of 16S rRNA in E. coli cells. Strong fluorescence was observed only when the phosphorothioate and quenched dabsyl probes bind side-by-side on a 16S rRNA target. The results demonstrate the use of QUAL probes to detect specific RNA sequences in bacterial cells without enzymes and without washing steps.

    View details for PubMedID 12903135

  • Efficient and simple solid-phase synthesis of short cyclic oligodeoxynucleotides bearing a phosphorothioate linkage ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Smietana, M., Kool, E. T. 2002; 41 (19): 3704-3707

    View details for Web of Science ID 000178609900044

    View details for PubMedID 12370937

  • Asymmetric recognition of DNA local distortion - Structure-based functional studies of eukaryotic Msh2-Msh6 JOURNAL OF BIOLOGICAL CHEMISTRY Drotschmann, K., Yang, W., Brownewell, F. E., Kool, E. T., Kunkel, T. A. 2001; 276 (49): 46225-46229


    Crystal structures of bacterial MutS homodimers bound to mismatched DNA reveal asymmetric interactions of the two subunits with DNA. A phenylalanine and glutamate of one subunit make mismatched base-specific interactions, and residues of both subunits contact the DNA backbone surrounding the mismatched base, but asymmetrically. A number of amino acids in MutS that contact the DNA are conserved in the eukaryotic Msh2-Msh6 heterodimer. We report here that yeast strains with amino acids substituted for residues inferred to interact with the DNA backbone or mismatched base have elevated spontaneous mutation rates consistent with defective mismatch repair. Purified Msh2-Msh6 with substitutions in the conserved Phe(337) and Glu(339) in Msh6 thought to stack or hydrogen bond, respectively, with the mismatched base do have reduced DNA binding affinity but normal ATPase activity. Moreover, wild-type Msh2-Msh6 binds with lower affinity to mismatches with thymine replaced by difluorotoluene, which lacks the ability to hydrogen bond. The results suggest that yeast Msh2-Msh6 interacts asymmetrically with the DNA through base-specific stacking and hydrogen bonding interactions and backbone contacts. The importance of these contacts decreases with increasing distance from the mismatch, implying that interactions at and near the mismatch are important for binding in a kinked DNA conformation.

    View details for Web of Science ID 000172573100100

    View details for PubMedID 11641390

  • The Phe-X-Glu DNA binding motif of MutS - The role of hydrogen bonding in mismatch recognition JOURNAL OF BIOLOGICAL CHEMISTRY Schofield, M. J., Brownewell, F. E., Nayak, S., Du, C. W., Kool, E. T., Hsieh, P. 2001; 276 (49): 45505-45508


    The crystal structures of MutS protein from Thermus aquaticus and Escherichia coli in a complex with a mismatch-containing DNA duplex reveal that the Glu residue in a conserved Phe-X-Glu motif participates in a hydrogen-bonded contact with either an unpaired thymidine or the thymidine of a G-T base-base mismatch. Here, the role of hydrogen bonding in mismatch recognition by MutS is assessed. The relative affinities of MutS for DNA duplexes containing nonpolar shape mimics of A and T, 4-methylbenzimidazole (Z), and difluorotoluene (F), respectively, that lack hydrogen bonding donors and acceptors, are determined in gel mobility shift assays. The results provide support for an induced fit mode of mismatch binding in which duplexes destabilized by mismatches are preferred substrates for kinking by MutS. Hydrogen bonding between the O epsilon 2 group of Glu and the mismatched base contributes only marginally to mismatch recognition and is significantly less important than the aromatic ring stack with the conserved Phe residue. A MutS protein in which Ala is substituted for Glu(38) is shown to be defective for mismatch repair in vivo. DNA binding studies reveal a novel role for the conserved Glu residue in the establishment of mismatch discrimination by MutS.

    View details for Web of Science ID 000172573100003

    View details for PubMedID 11602569

  • Significance of nucleobase shape complementarity and hydrogen bonding in the formation and stability of the closed polymerase-DNA complex BIOCHEMISTRY Dzantiev, L., Alekseyev, Y. O., Morales, J. C., Kool, E. T., Romano, L. J. 2001; 40 (10): 3215-3221


    DNA polymerases insert a dNTP by a multistep mechanism that involves a conformational rearrangement from an open to a closed ternary complex, a process that positions the incoming dNTP in the proper orientation for phosphodiester bond formation. In this work, the importance and relative contribution of hydrogen-bonding interactions and the geometric shape of the base pair that forms during this process were studied using Escherichia coli DNA polymerase I (Klenow fragment, 3'-exonuclease deficient) and natural dNTPs or non-hydrogen-bonding dNTP analogues. Both the geometric fit of the incoming nucleotide and its ability to form Watson-Crick hydrogen bonds with the template were found to contribute to the stability of the closed ternary complex. Although the formation of a closed complex in the presence of a non-hydrogen-bonding nucleotide analogue could be detected by limited proteolysis analysis, a comparison of the stabilities of the ternary complexes indicated that hydrogen-bonding interactions between the incoming dNTP and the template increase the stability of the complex by 6-20-fold. Any deviation from the Watson-Crick base pair geometry was shown to have a destabilizing effect on the closed complex. This degree of destabilization varied from 3- to 730-fold and was found to be correlated with the size of the mismatched base pair. Finally, a stable closed complex is not formed in the presence of a ddNTP or rNTP. These results are discussed in relation to the steric exclusion model for the nucleotide insertion.

    View details for Web of Science ID 000167407000025

    View details for PubMedID 11258938

  • Nonenzymatic autoligation in direct three-color detection of RNA and DNA point mutations NATURE BIOTECHNOLOGY Xu, Y. Z., Karalkar, N. B., Kool, E. T. 2001; 19 (2): 148-152


    Enzymatic ligation methods are useful in diagnostic detection of DNA sequences. Here we describe the investigation of nonenzymatic phosphorothioate-iodide DNA autoligation chemistry as a method for detection and identification of both RNA and DNA sequences. Combining ligation specificity with the hybridization specificity of the ligated product is shown to yield discrimination of a point mutation as high as >10(4)-fold. Unlike enzymatic ligations, this reaction is found to be equally efficient on RNA or DNA templates. The reaction is also shown to exhibit a significant level of self-amplification, with the template acting in catalytic fashion to ligate multiple pairs of probes. A strategy for fluorescence labeling of three autoligating energy transfer (ALET) probes and directly competing them for autoligation on a target sequence is described. The method is tested in several formats, including solution phase, gel, and blot assays. The ALET probe design offers direct RNA detection, combining high sequence specificity with an easily detectable color change by fluorescence resonance energy transfer (FRET).

    View details for Web of Science ID 000166754700021

    View details for PubMedID 11175729

  • Hydrogen bonding, base stacking, and steric effects in DNA replication ANNUAL REVIEW OF BIOPHYSICS AND BIOMOLECULAR STRUCTURE Kool, E. T. 2001; 30: 1-22


    Understanding the mechanisms by which genetic information is replicated is important both to basic knowledge of biological organisms and to many useful applications in biomedical research and biotechnology. One of the main functions of a DNA polymerase enzyme is to help DNA recognize itself with high specificity when a strand is being copied. Recent studies have shed new light on the question of what physical forces cause a polymerase enzyme to insert a nucleotide into a strand of DNA and to choose the correct nucleotide over the incorrect ones. This is discussed in the light of three main forces that govern DNA recognition: base stacking, Watson-Crick hydrogen bonding, and steric interactions. These factors are studied with natural and structurally altered DNA nucleosides.

    View details for Web of Science ID 000169531600002

    View details for PubMedID 11340050

  • Synthetically modified DNAs as substrates for polymerases CURRENT OPINION IN CHEMICAL BIOLOGY Kool, E. T. 2000; 4 (6): 602-608


    DNA polymerase enzymes process their natural substrates with very high specificity. Yet recent experiments have shown that these enzymes can also process DNA in which the backbone or bases are modified to a surprising degree. Such experiments have important implications in understanding the mechanisms of DNA replication, and suggest important biotechnological uses as well.

    View details for Web of Science ID 000165854000002

    View details for PubMedID 11102863

  • Functional hydrogen-bonding map of the minor groove binding tracks of six DNA polymerases BIOCHEMISTRY Morales, J. C., Kool, E. T. 2000; 39 (42): 12979-12988


    Recent studies have identified amino acid side chains forming several hydrogen bonds in the DNA minor groove as potentially important in polymerase replication of DNA. Few studies have probed these interactions on the DNA itself. Using non-hydrogen-bonding nucleoside isosteres, we have now studied effects in both primer and template strands with several polymerases to investigate the general importance of these interactions. All six polymerases show differences in the H-bonding effects in the minor groove. Two broad classes of activity are seen, with a first group of DNA polymerases (KF(-), Taq, and HIV-RT) that efficiently extends nonpolar base pairs containing nucleoside Q (9-methyl-1H-imidazo[4,5-b]pyridine) but not the analogue Z (4-methylbenzimidazole), implicating a specific minor groove interaction at the first extension site. A second group of polymerases (Pol alpha, Pol beta, and T7(-)) fails to extend all non-H-bonding base pairs, indicating that these enzymes may need minor groove hydrogen bonds at both minor groove sites or that they are especially sensitive to noncanonical DNA structure or stability. All DNA polymerases examined use energetically important minor groove interactions to probe newly synthesized base pairs before extending them. The positions of these interactions vary among the enzymes, and only a subset of the interactions identified structurally appears to be functionally important. In addition, polymerases appear to be differently sensitive to small changes in base pair geometry.

    View details for DOI 10.1021/bi001578o

    View details for Web of Science ID 000090107300026

    View details for PubMedID 11041863

  • Solution structure of a nonpolar, non-hydrogen-bonded base pair surrogate in DNA JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Guckian, K. M., Krugh, T. R., Kool, E. T. 2000; 122 (29): 6841-6847
  • Factors contributing to aromatic stacking in water: Evaluation in the context of DNA JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Guckian, K. M., Schweitzer, B. A., Ren, R. X., Sheils, C. J., Tahmassebi, D. C., Kool, E. T. 2000; 122 (10): 2213-2222
  • Importance of terminal base pair hydrogen-bonding in 3 '-end proofreading by the Klenow fragment of DNA polymerase I BIOCHEMISTRY Morales, J. C., Kool, E. T. 2000; 39 (10): 2626-2632


    We describe studies aimed at evaluating the physical factors governing the rate of 3'-end proofreading by the Klenow fragment of E. coli DNA polymerase I. Two nonpolar deoxynucleoside isosteres containing 2,4-difluorotoluene (F) and 4-methylbenzimidazole (Z), which are non-hydrogen-bonding shape mimics of thymine and adenine, respectively, are used to investigate the effects of base pair geometry and stability on the rate of this exonuclease activity. Steady-state kinetics measurements show that complementary T.A base pairs at the end of a primer-template duplex are edited 14-40-fold more slowly than mismatches. By contrast, a 3'-end T residue in a T. Z pair is edited at a rate equivalent to that of natural base mismatches despite the fact that it resembles a T.A pair in structure. Similarly, the A in an A.F pair is edited as rapidly as a mismatched pair despite its close structural mimicry of an A.T pair. Interestingly, when the base pairs are reversed and F or Z is located at the 3'-end, they are edited more slowly, possibly implicating specific interactions between the exonuclease domain and the base of the nucleotide being edited. Finally, thermal denaturation studies are carried out to investigate the relationship between editing and the ease of unwinding of the duplex. The rapid editing of bases opposite F or Z residues at the duplex terminus seems to correlate well with the stability of these base pairs when placed in a context resembling a primer-template duplex. In general, the rate of 3'-end editing appears to be governed by the rate of fraying of the DNA terminal pair, and base pair geometry appears to have little effect.

    View details for Web of Science ID 000085905300021

    View details for PubMedID 10704212

  • Varied molecular interactions at the active sites of several DNA polymerases: Nonpolar nucleoside isosteres as probes JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Morales, J. C., Kool, E. T. 2000; 122 (6): 1001-1007
  • The virtues of self-binding: high sequence specificity for RNA cleavage by self-processed hammerhead ribozymes NUCLEIC ACIDS RESEARCH Ohmichi, T., Kool, E. T. 2000; 28 (3): 776-783


    Naturally occurring hammerhead ribozymes are produced by rolling circle replication followed by self-cleavage. This results in monomer-length catalytic RNAs which have self-complementary sequences that can occupy their trans -binding domains and potentially block their ability to cleave other RNA strands. Here we show, using small self-processed ribozymes, that this self-binding does not necessarily inhibit trans -cleavage and can result in greatly elevated discrimination against mismatches. We utilized a designed 63 nt circular DNA to encode the synthesis of a self-processed ribozyme, MDR63. Rolling circle transcription followed by self-processing produced the desired 63 nt ribozyme, which potentially can bind mdr-1 RNA with 9+9 nt of complementarity or bind itself with 4+5 nt of self-complementarity by folding back its ends to form hairpins. Kinetics of trans -cleavage of short complementary and mismatched RNAs were measured under multiple turnover conditions, in comparison to a standard 40 nt ribozyme (MDR40) that lacks the self-complementary ends. The results show that MDR63 cleaves an mdr-1 RNA target with a k (cat)/ K (m)almost the same as MDR40, but with discrimination against mismatches up to 20 times greater. Based on folding predictions, a second self-processed ribozyme (UG63) having a single point mutation was synthesized; this displays even higher specificity (up to 100-fold) against mismatches. The results suggest that self-binding ends may be generally useful for increasing sequence specificity of ribozymes.

    View details for Web of Science ID 000085147000015

    View details for PubMedID 10637330

  • Roles of Watson-Crick and minor groove hydrogen bonds in DNA replication COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY Kool, E. T. 2000; 65: 93-102

    View details for Web of Science ID 000169676800011

    View details for PubMedID 12760024

  • Mimicking the structure and function of DNA: Insights into DNA stability and replication ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Kool, E. T., Morales, J. C., Guckian, K. M. 2000; 39 (6): 990-1009
  • Polymerase activities and RNA structures in the atomic force microscope JOURNAL OF STRUCTURAL BIOLOGY HANSMA, H. G., Golan, R., Hsieh, W., Daubendiek, S. L., Kool, E. T. 1999; 127 (3): 240-247


    The structures of the reaction products are the basis for novel polymerase assays using the atomic force microscope (AFM). Polymerases are the enzymes involved in transcription and replication of DNA. Rapid semiquantitative estimates of the activity of DNA polymerases such as Sequenase, Taq polymerase, and AMV reverse transcriptase and RNA polymerases (RNAP) such as Escherichia coli RNAP were obtained from AFM images of the nucleic acids after polymerase reactions. DNA polymerases were assayed via replication of the single-stranded φX-174 virion. RNAP was assayed via transcription, using a rolling circle DNA template that produces long strands of RNA. In some cases, AFM was better than agarose gel electrophoresis for assaying DNA polymerase activity, since aggregation prevented the DNA from entering the agarose gel. Extended molecules of single-stranded RNA synthesized with the rolling circle DNA template showed varied conformations and degrees of stretching. Some structural differences were observed between two RNAs-a ribozyme concatamer and an RNA with 90% purines.

    View details for Web of Science ID 000083736600005

    View details for PubMedID 10544049

  • Minor groove interactions between polymerase and DNA: More essential to replication than Watson-Crick hydrogen bonds? JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Morales, J. C., Kool, E. T. 1999; 121 (10): 2323-2324
  • Oriented, active Escherichia coli RNA polymerase: An atomic force microscope study BIOPHYSICAL JOURNAL Thomson, N. H., Smith, B. L., Almqvist, N., Schmitt, L., Kashlev, M., Kool, E. T., Hansma, P. K. 1999; 76 (2): 1024-1033


    Combining a system for binding proteins to surfaces (Sigal, G. B., C. Bamdad, A. Barberis, J. Strominger, and G. M. Whitesides. 1996. Anal. Chem. 68:490-497) with a method for making ultraflat gold surfaces (Hegner, M., P. Wagner, and G. Semenza. 1993. Surface Sci. 291:39-46 1993) has enabled single, oriented, active Escherichia coli RNA polymerase (RNAP) molecules to be imaged under aqueous buffer using tapping-mode atomic force microscopy (AFM). Recombinant RNAP molecules containing histidine tags (hisRNAP) on the C-terminus were specifically immobilized on ultraflat gold via a mixed monolayer of two different omega-functionalized alkanethiols. One alkanethiol was terminated in an ethylene-glycol (EG) group, which resists protein adsorption, and the other was terminated in an N-nitrilotriacetic acid (NTA) group, which binds the histidine tag through two coordination sites with a nickel ion. AFM images showed that these two alkanethiols phase-segregate. Specific binding of the hisRNAP molecules was followed in situ by injecting proteins directly into the AFM fluid cell. The activity of the hisRNAP bound to the NTA groups was confirmed with a 42-base circular single-stranded DNA template (rolling circle), which the RNAP uses to produce huge RNA transcripts. These transcripts were imaged in air after the samples were rinsed and dried, since RNA also has low affinity for the EG-thiol and cannot be imaged under the buffers we used.

    View details for Web of Science ID 000078368800041

    View details for PubMedID 9916034

  • Novel nucleoside analogues with fluorophores replacing the DNA base HELVETICA CHIMICA ACTA Strassler, C., DAVIS, N. E., Kool, E. T. 1999; 82 (12): 2160-2171


    We describe the preparation and fluorescence properties of a set of new nucleosides in which a known hydrocarbon or oligothiophene fluorophore replaces the DNA base at C(1) of the deoxyribose moiety (see 3a - f). These compounds are potentially useful as probes in the study of the structure and dynamics of nucleic acids and their complexes with proteins. In addition, they may find use as fluorescent labels for nucleic-acid-based biomedical diagnostics methods. The fluorophores conjugated to deoxyribose at C(1) in the α-d-form include terphenyl, stilbene, terthiophene, benzoterthiophene, and pyrene. Also included is a non-fluorescent spacer in which cyclohexene replaces the DNA base. The nucleosides are derived from brominated fluorophore precursors and Hoffer's 2-deoxy-3,5-di-O-(p-toluoyl)-d-ribofuranosyl chloride. The emission maxima of the free nucleosides range from 345 to 536 nm. Also described are the 5'-(dimethoxytrityl) 3'-O-phosphoramidite derivatives 5a - f, suitable for incorporation into oligonucleotides by automated synthesizers.

    View details for Web of Science ID 000084557400009

  • Tightening the belt on polymerases: Evaluating the physical constraints on enzyme substrate size ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Frieden, M., Pedroso, E., Kool, E. T. 1999; 38 (24): 3654-3657
  • Structure and base pairing properties of a replicable nonpolar isostere for deoxyadenosine JOURNAL OF ORGANIC CHEMISTRY Guckian, K. M., Morales, J. C., Kool, E. T. 1998; 63 (26): 9652-9656
  • Generation of RNA ladders by rolling circle transcription of small circular oligodeoxyribonucleotides BIOTECHNIQUES Diegelman, A. M., Daubendiek, S. L., Kool, E. T. 1998; 25 (5): 754-?

    View details for Web of Science ID 000076896100001

    View details for PubMedID 9821573

  • Probing DNA sequences in solution with a monomer-excimer fluorescence color change NUCLEIC ACIDS RESEARCH Paris, P. L., Langenhan, J. M., Kool, E. T. 1998; 26 (16): 3789-3793


    The use of a simple fluorescent nucleoside analogue in detection of point mutations by hybridization in solution is described. Pyrene is placed at 3' and 5' ends of a pair of oligodeoxynucleotide probes via a phosphoramidite derivative of deoxyribose with this fluorophore attached at the 1' position, replacing a DNA base. Adjacent binding of dual probes containing this fluorophore to a complementary target sequence results in a pronounced spectral change from blue pyrene monomer emission (lambdamax= 381 398 nm) to green-white excimer emission (lambdamax= 490 nm). Optimization of the relative binding positions of the two probes shows that the greatest spectral change occurs when they bind with partial end overlap. In optimum orientation, the monomer emission band for the probes decreases intensity by as much as a factor of seven and the excimer band increases up to 40-fold on binding a complementary target. Application to the detection of a single-base point mutation in solution is described.

    View details for Web of Science ID 000075408200024

    View details for PubMedID 9685497

  • A thymidine triphosphate shape analog lacking Watson-Crick pairing ability is replicated with high sequence selectivity PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Moran, S., Ren, R. X., Kool, E. T. 1997; 94 (20): 10506-10511


    Compound 1 (F), a nonpolar nucleoside analog that is isosteric with thymidine, has been proposed as a probe for the importance of hydrogen bonds in biological systems. Consistent with its lack of strong H-bond donors or acceptors, F is shown here by thermal denaturation studies to pair very poorly and with no significant selectivity among natural bases in DNA oligonucleotides. We report the synthesis of the 5'-triphosphate derivative of 1 and the study of its ability to be inserted into replicating DNA strands by the Klenow fragment (KF, exo- mutant) of Escherichia coli DNA polymerase I. We find that this nucleotide derivative (dFTP) is a surprisingly good substrate for KF; steady-state measurements indicate it is inserted into a template opposite adenine with efficiency (Vmax/Km) only 40-fold lower than dTTP. Moreover, it is inserted opposite A (relative to C, G, or T) with selectivity nearly as high as that observed for dTTP. Elongation of the strand past F in an F-A pair is associated with a brief pause, whereas that beyond A in the inverted A-F pair is not. Combined with data from studies with F in the template strand, the results show that KF can efficiently replicate a base pair (A-F/F-A) that is inherently very unstable, and the replication occurs with very high fidelity despite a lack of inherent base-pairing selectivity. The results suggest that hydrogen bonds may be less important in the fidelity of replication than commonly believed and that nucleotide/template shape complementarity may play a more important role than previously believed.

    View details for Web of Science ID A1997XY99800007

    View details for PubMedID 9380669

  • C-nucleosides derived from simple aromatic hydrocarbons SYNLETT CHAUDHURI, N. C., Ren, R. X., Kool, E. T. 1997: 341-?
  • Generation of catalytic RNAs by rolling transcription of synthetic DNA nanocircles NATURE BIOTECHNOLOGY Daubendiek, S. L., Kool, E. T. 1997; 15 (3): 273-277


    Small catalytic RNAs are commonly produced either by transcription of promoter-driven linear DNA templates or by stepwise chemical synthesis on solid supports. We describe a different approach, in which very small chemically synthesized circular DNAs serve as efficient templates for generation of catalytic RNAs in vitro. The circles are 83 nucleotides in size, are single stranded, and contain no canonical RNA polymerase promoters. Despite this, T7 and Escherichia coli RNA polymerases transcribe the circles by a rolling mechanism, producing long concatemeric RNAs (approximately 7,500 nt). During the transcription reaction, the repeating RNAs self-cleave, ultimately reaching monomer length. Despite having self-complementary sequences at their substrate-binding domains, these monomeric 83-nt RNAs are shown to be catalytically active ribozymes that sequence-specifically cleave RNA targets in trans. In addition, a circular vector encoding a repeating (non-self-processing) ribozyme is described; the resulting multimeric ribozyme, targeted to a sequence in the HIV-1 genome, is also catalytically active in trans. This novel approach to the synthesis of catalytic RNAs offers a number of differences and potential advantages over current approaches to RNA synthesis.

    View details for Web of Science ID A1997WM05000033

    View details for PubMedID 9062929

  • Difluorotoluene, a nonpolar isostere for thymine, codes specifically and efficiently for adenine in DNA replication JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Moran, S., Ren, R. X., RUMNEY, S., Kool, E. T. 1997; 119 (8): 2056-2057
  • Escherichia coli RNA polymerase activity observed using atomic force microscopy BIOCHEMISTRY Kasas, S., Thomson, N. H., Smith, B. L., HANSMA, H. G., Zhu, X. S., Guthold, M., Bustamante, C., Kool, E. T., Kashlev, M., Hansma, P. K. 1997; 36 (3): 461-468


    Fluid tapping-mode atomic force microscopy (AFM) was used to observe Escherichia coli RNA polymerase (RNAP) transcribing two different linear double-stranded (ds) DNA templates. The transcription process was detected by observing the translocation of the DNA template by RNAP on addition of ribonucleoside 5'-triphosphates (NTPs) in sequential AFM images. Stalled ternary complexes of RNAP, dsDNA and nascent RNA were adsorbed onto a mica surface and imaged under continuously flowing buffer. On introduction of all four NTPs, we observed some DNA molecules being pulled through the RNAP, some dissociating from the RNAP and others which did not move relative to the RNAP. The transcription rates were observed to be approximately 0.5-2 bases/s at our NTP concentrations, approximately 5 microM. The RNA transcripts were not unambiguously imaged in fluid. However, in experiments using a small single-stranded (ss) circular DNA template, known as a rolling circle, transcripts up to 1 or 2 microns long could be observed with tapping mode AFM once the samples were dried and imaged in air. This confirmed our observations of the transcriptional activity of RNAP adsorbed onto mica. This work illustrates that the development of tapping-mode in fluid has made it possible to use AFM to follow biological processes at the molecular level and get new insights about the variability of activity of individual molecules bound to a surface.

    View details for Web of Science ID A1997WD69800001

    View details for PubMedID 9012661

  • Highly precise shape mimicry by a difluorotoluene deoxynucleoside, a replication-competent substitute for thymidine ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Guckian, K. M., Kool, E. T. 1997; 36 (24): 2825-2828
  • Experimental measurement of aromatic stacking affinities in the context of duplex DNA JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Guckian, K. M., Schweitzer, B. A., Ren, R. X., Sheils, C. J., Paris, P. L., Tahmassebi, D. C., Kool, E. T. 1996; 118 (34): 8182-8183
  • Naphthalene, phenanthrene, and pyrene as DNA base analogues: Synthesis, structure, and fluorescence in DNA JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Ren, R. X., CHAUDHURI, N. C., Paris, P. L., RUMNEY, S., Kool, E. T. 1996; 118 (33): 7671-7678
  • Non-hydrogen bonding 'terminator' nucleosides increase the 3'-end homogeneity of enzymatic RNA and DNA synthesis NUCLEIC ACIDS RESEARCH Moran, S., Ren, R. X., Sheils, C. J., RUMNEY, S., Kool, E. T. 1996; 24 (11): 2044-2052


    We report the use of novel non-polar nucleoside analogues as terminators of enzymatic RNA and DNA synthesis. Standard 'runoff' RNA synthesis by T7 RNA polymerase gives RNA products which have ragged ends as a result of transcription which often extends beyond the end of the template DNA strand. Similarly, the Klenow fragment of Escherichia coli DNA polymerase I tends to run past the end of the template strand during DNA synthesis. We report here that certain non-hydrogen-bonding nucleoside analogues, when placed at the downstream 5'-end of a template DNA strand, cause the polymerases to stop more abruptly at the last coding nucleotide. This results in a considerably more homogeneous oligonucleotide being produced. Three novel nucleosides are tested as potential terminators: 4-methylindole beta-deoxynucleoside (M), 1-naphthyl alpha-deoxynucleoside (N) and 1-pyrenyl alpha-deoxynucleoside (P). Comparison is made to an abasic nucleoside (phi) and to unterminated synthesis. Of these, M is found to be the most efficient at terminating transcription, and both P and M are highly effective at terminating DNA synthesis. It is also found that the ability of a nucleoside to stall synthesis when it is internally placed in the template strand is not necessarily a good predictor of terminating ability at the end of a template. Such terminator nucleosides may be useful in the preparative enzymatic synthesis of RNA and DNA, rendering purification simpler and lowering the cost of synthesis by preventing the uptake of potentially costly nucleotides into unwanted products.

    View details for Web of Science ID A1996UR37900010

    View details for PubMedID 8668534

  • Formation of stable DNA loops by incorporation of nonpolar, non-hydrogen-bonding nucleoside isosteres ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Ren, X. F., Schweitzer, B. A., Sheils, C. J., Kool, E. T. 1996; 35 (7): 743-746
  • Rolling circle DNA synthesis: Small circular oligonucleotides as efficient templates for DNA polymerases JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Liu, D. Y., Daubendiek, S. L., ZILLMAN, M. A., Ryan, K., Kool, E. T. 1996; 118 (7): 1587-1594

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