This is a renewal application for a research program that focuses on the development and application of deoxyribozymes for bioorganic chemistry. Deoxyribozymes (DNA enzymes) are DNA molecules that have particular catalytic activities. Long-term objectives of this research are to expand the limits of DNA catalysis by deoxyribozymes and to understand and control this catalysis. Achieving these goals should enable practical application of deoxyribozymes, which is another long-term objective of this research. Nature uses ribozymes (RNA enzymes) to catalyze biologically relevant reactions such as protein synthesis in the ribosome and RNA splicing in the spliceosome. Many other natural catalytic RNAs have been identified. Although no natural deoxyribozymes are known, the chemical properties of DNA suggest that it has catalytic potential when in largely single-stranded form. We and others have shown that catalytically active DNA sequences can be identified by in vitro selection from large pools of random DNA sequences. We hypothesize that DNA can catalyze reactions not only of oligonucleotide substrates (as explored extensively by us and others) but also small molecules and proteins. We propose to test this hypothesis by systematic in vitro selection experiments designed to probe the limits of DNA catalysis. We also propose to use a variety of approaches to characterize a specific deoxyribozyme that we previously identified and to apply this deoxyribozyme to address several fundamental biochemical questions related to RNA.
Aim 1 systematically explores small molecules as deoxyribozyme substrates. This is a key step in applying DNA more widely to small-molecule bioorganic chemistry.
Aim 2 seeks deoxyribozymes that catalyze reactions of amino acid sidechains, which will improve our understanding of how to achieve DNA-catalyzed modifications of proteins.
Aim 3 targets cleavage of peptide linkages as a specific example of DNA catalysis applied to protein substrates. Practical downstream applications of protease deoxyribozymes include (a) as an alternative to trypsin and other proteases in proteomics for generation of large protein fragments without further degradation, and (b) as an approach for in vivo zymogen activation, such as formation of coagulation factor VIIa as a novel approach to treatment of bleeding.
Aim 4 is directed towards detailed biophysical and structural characterizations of a deoxyribozyme that creates branched RNA, which is the natural intermediate of RNA splicing. These studies will increase our fundamental understanding of nucleic acid catalysis, which is crucial if we are to improve deoxyribozyme function and incorporate rational design elements alongside in vitro selection. Finally, Aim 5 applies deoxyribozymes to three important biochemical problems that involve or use branched RNA. We anticipate that all of these investigations will broaden our basic understanding of the catalytic potential of DNA as well as expand the practical biochemical utility of deoxyribozymes.

Public Health Relevance

Catalytic DNA molecules (deoxyribozymes) are an intriguing new form of catalyst that can be used for increasing our basic understanding of nucleic acids and for practical applications. The proposed research expands the chemical scope of DNA catalysis and applies deoxyribozymes to fundamental biochemical questions involving RNA, which plays a central role throughout biology.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Macromolecular Structure and Function E Study Section (MSFE)
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Preusch, Peter C
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University of Illinois Urbana-Champaign
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Lee, Yujeong; Klauser, Paul C; Brandsen, Benjamin M et al. (2017) DNA-Catalyzed DNA Cleavage by a Radical Pathway with Well-Defined Products. J Am Chem Soc 139:255-261
Silverman, Scott K (2016) Catalytic DNA: Scope, Applications, and Biochemistry of Deoxyribozymes. Trends Biochem Sci 41:595-609
Hesser, Anthony R; Brandsen, Benjamin M; Walsh, Shannon M et al. (2016) DNA-catalyzed glycosylation using aryl glycoside donors. Chem Commun (Camb) 52:9259-62
Camden, Alison J; Walsh, Shannon M; Suk, Sarah H et al. (2016) DNA Oligonucleotide 3'-Phosphorylation by a DNA Enzyme. Biochemistry 55:2671-6
Chu, Chih-Chi; Silverman, Scott K (2016) Assessing histidine tags for recruiting deoxyribozymes to catalyze peptide and protein modification reactions. Org Biomol Chem 14:4697-703
Wang, Puzhou; Silverman, Scott K (2016) DNA-Catalyzed Introduction of Azide at Tyrosine for Peptide Modification. Angew Chem Int Ed Engl 55:10052-6
Zhou, Cong; Avins, Joshua L; Klauser, Paul C et al. (2016) DNA-Catalyzed Amide Hydrolysis. J Am Chem Soc 138:2106-9
Walsh, Shannon M; Konecki, Stephanie N; Silverman, Scott K (2015) Identification of Sequence-Selective Tyrosine Kinase Deoxyribozymes. J Mol Evol 81:218-24
Silverman, Scott K (2015) Pursuing DNA catalysts for protein modification. Acc Chem Res 48:1369-79
Chandrasekar, Jagadeeswaran; Wylder, Adam C; Silverman, Scott K (2015) Phosphoserine Lyase Deoxyribozymes: DNA-Catalyzed Formation of Dehydroalanine Residues in Peptides. J Am Chem Soc 137:9575-8

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