The proposed research concerns the in vitro evolution of novel nucleic acid enzymes that can be used to modulate biological processes. Nucleic adds have both genetic and catalytic properties, making it straightforward to couple amplification and mutation of their genetic sequence with selection based on their corresponding catalytic properties. Efforts will focus on the development of two classes of catalytic nucleic acids: DNA enzymes with N-glycosylase activity and RNA enzymes with homing endoribonudease activity. The former will be evolved to repair specific lesions of DNA that result from mutation or oxidative damage. The latter will be constructed by joining two existing RNA enzymes to create a bifunctional molecule that can insert itself in an irreversible manner at a specific location within RNA. It will be used to perform targeted gene disruption or targeted insertion of a coding region within mRNA. Attention also will be directed toward understanding the evolution process itself. Comparisons will be made among molecules that arise as a consequence of different degrees of selection pressure or varying complexity of their component subunits. A new approach employing a quench-flow device will be used to evolve ENA enzymes with very fast reaction rates. RNA enzymes also will be developed that operate under conditions of extreme pH or temperature, shedding light on the limits of RNA-based catalytic function. Finally, a new class of tethered small-molecule cofactors will be synthesized and supplied to the nucleic add enzymes to assist in their catalytic function. These cofactors will consist of either an amino add or a short peptide that is attached to the end of an oligodeoxynucleotide adapter. The adapter will allow the cofactor to be bound readily by the enzyme, allowing evolution to exploit the bound cofactor for use in catalysis.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM065130-01
Application #
6457561
Study Section
Bio-Organic and Natural Products Chemistry Study Section (BNP)
Program Officer
Ikeda, Richard A
Project Start
2002-04-01
Project End
2006-03-31
Budget Start
2002-04-01
Budget End
2003-03-31
Support Year
1
Fiscal Year
2002
Total Cost
$333,066
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Olea, Charles; Joyce, Gerald F (2016) Real-Time Detection of a Self-Replicating RNA Enzyme. Molecules 21:
Joyce, Gerald F (2015) Reflections of a Darwinian Engineer. J Mol Evol 81:146-9
Olea Jr, Charles; Weidmann, Joachim; Dawson, Philip E et al. (2015) An L-RNA Aptamer that Binds and Inhibits RNase. Chem Biol 22:1437-1441
Sczepanski, Jonathan T; Joyce, Gerald F (2015) Specific Inhibition of MicroRNA Processing Using L-RNA Aptamers. J Am Chem Soc 137:16032-7
Olea Jr, Charles; Joyce, Gerald F (2015) Ligand-dependent exponential amplification of self-replicating RNA enzymes. Methods Enzymol 550:23-39
Breaker, Ronald R; Joyce, Gerald F (2014) The expanding view of RNA and DNA function. Chem Biol 21:1059-65
Petrie, Katherine L; Joyce, Gerald F (2014) Limits of neutral drift: lessons from the in vitro evolution of two ribozymes. J Mol Evol 79:75-90
Robertson, Michael P; Joyce, Gerald F (2014) Highly efficient self-replicating RNA enzymes. Chem Biol 21:238-45
Sczepanski, Jonathan T; Joyce, Gerald F (2013) Binding of a structured D-RNA molecule by an L-RNA aptamer. J Am Chem Soc 135:13290-3
Ferretti, Antonio C; Joyce, Gerald F (2013) Kinetic properties of an RNA enzyme that undergoes self-sustained exponential amplification. Biochemistry 52:1227-35

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