Nonpolar nucleoside isosteres have proven widely useful in the study of electrostatic and steric interactions between DNA and proteins. In the past four years they have been employed in over a dozen laboratories as biomolecular probes, and have revealed new insights into electrostatic and steric effects in DNA polymerase active sites, in electrostatic interactions in DNA bending, in replication machinery in vivo, and in a number of DNA repair systems as well. These molecular analogs are important because they are perhaps the best available chemical tools that allow for the separation of steric effects from electrostatic effects in nucleic acid recognition. Our long-term goals are to use this class of compounds more widely as basic tools to analyze mechanism and function in protein-nucleic acid interactions. The overarching theme is specificity of protein-nucleic acid recognition. To explore this topic we plan to develop a broader set of analogs with varied size and shape, and sets that can be applied in RNA as well as DNA systems. Moreover, we hope to extend our studies to pathways in living cells, to gain a more complex understanding of interactions beyond simple in vitro systems. Polymerase enzymes of particular interest are the low-fidelity repair polymerases pol iota and Dpo4, as well as three diverse reverse transcriptases: telomerase, Ty3, and HIV-RT. One chief focus is the testing of our """"""""active site tightness"""""""" hypothesis for fidelity of replication. The fidelity of genome replication depends not only on polymerases but on repair enzymes as well, and we plan new studies of two enzymes that repair oxidative DNA damage. Finally, the specificity of RNA interactions are of increasing interest, especially those occurring in RNA interference mechanisms. Over the near term covered by this proposal, our specific aims are to (1) investigate active site tightness effects in DNA polymerase fidelity with new analogs of increasing size; (2) carry out in vivo replication studies of nonpolar nucleoside mimics, focusing on steric and minor groove effects; (3) study active site interactions of three diverse reverse transcriptases; (4) conduct new mechanistic studies of two DNA base excision repair enzymes; and (5) perform an investigation of newly discovered asymmetry in double-stranded RNA interference mechanisms.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Bio-Organic and Natural Products Chemistry Study Section (BNP)
Program Officer
Fabian, Miles
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Stanford University
Schools of Arts and Sciences
United States
Zip Code
Hwang, Candy S; Xu, Liang; Wang, Wei et al. (2016) Functional interplay between NTP leaving group and base pair recognition during RNA polymerase II nucleotide incorporation revealed by methylene substitution. Nucleic Acids Res 44:3820-8
Xu, Liang; Butler, Kyle Vincent; Chong, Jenny et al. (2014) Dissecting the chemical interactions and substrate structural signatures governing RNA polymerase II trigger loop closure by synthetic nucleic acid analogues. Nucleic Acids Res 42:5863-70
Xu, Liang; Da, Linati; Plouffe, Steven W et al. (2014) Molecular basis of transcriptional fidelity and DNA lesion-induced transcriptional mutagenesis. DNA Repair (Amst) 19:71-83
Kool, Eric T; Crisalli, Pete; Chan, Ke Min (2014) Fast alpha nucleophiles: structures that undergo rapid hydrazone/oxime formation at neutral pH. Org Lett 16:1454-7
Crisalli, Pete; Kool, Eric T (2013) Importance of ortho proton donors in catalysis of hydrazone formation. Org Lett 15:1646-9
Crisalli, Pete; Kool, Eric T (2013) Water-soluble organocatalysts for hydrazone and oxime formation. J Org Chem 78:1184-9
Winnacker, Malte; Kool, Eric T (2013) Artificial genetic sets composed of size-expanded base pairs. Angew Chem Int Ed Engl 52:12498-508
Jung, Jong-Wha; Edwards, Sarah K; Kool, Eric T (2013) Selective fluorogenic chemosensors for distinct classes of nucleases. Chembiochem 14:440-4
Singh, Vijay; Wang, Shenliang; Kool, Eric T (2013) Genetically encoded multispectral labeling of proteins with polyfluorophores on a DNA backbone. J Am Chem Soc 135:6184-91
Spitale, Robert C; Crisalli, Pete; Flynn, Ryan A et al. (2013) RNA SHAPE analysis in living cells. Nat Chem Biol 9:18-20

Showing the most recent 10 out of 42 publications