We propose to study how biological information, in the form of specific nucleobases, is recognized and transferred accurately in cellular pathways. Errors in this information transfer lead to carcinogenesis and to drug resistance, and so understanding how they arise, and how to modulate them, can have direct implications in medical treatment of cancer and infectious disease. Our approach to studying these biological processes is to develop and study new DNA and RNA base analogs that modulate electrostatics and shape in a systematic way. We will use sets of these molecules to probe steric and electronic effects in four nucleobase-dependent processes, including the uptake and metabolism of specific nucleosides into cells;the polymerase-dependent copying of DNA and RNA sequences;protein-mediated RNA-RNA recognition in RNA interference;and specific recognition of damaged nucleobases. All of these require that the biological systems can accurately discriminate one nucleobase from others;
we aim to study how this occurs. During the recent funding period we have made strong progress in understanding how varied classes of DNA polymerases identify correct versus incorrect nucleotides. This has led to new hypotheses concerning active site rigidity and flexibility, and how repair enzymes differ from replicative ones. Moreover, using new RNA base analogs we have generated exciting preliminary data on how siRNAs recognize and downregulate specific mRNAs. Finally, we have developed new analogs of damaged DNA bases, and have begun to use them to study mutagenesis and repair. In the near term covered by this proposal, we plan to test several new hypotheses for nucleotide recognition.
Our specific aims i nclude (1) study of the origins of sequence recognition in RNA interference;(2) researching the mechanisms of kinase-mediated nucleotide uptake and metabolism;(3) investigation of mismatch and damage recognition by new classes of polymerases. We expect that this work will lead to important basic insights into the chemistry and biology of nucleobases, and possibly to new diagnostic and therapeutic strategies involving nucleosides and nucleic acids.

Public Health Relevance

We propose to study how the four nucleic acid bases of DNA and RNA are discriminated from one another in the biological pathways that involve this genetic information. This will be done by design and study of modified versions of these bases, and their function in these pathways. The work is important because it provides basic information about how mutations arise in DNA, and suggests new strategies for increasing activity and lowering toxicity of potential nucleotide- based drugs.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-BCMB-A (03))
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Fabian, Miles
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Stanford University
Schools of Arts and Sciences
United States
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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
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
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

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