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RNA-Cleaving DNA Enzymes with Altered Regio- or EnantioselectivityIn vitro evolution methods were used to obtain DNA enzymes that cleave either a 2',5' - phosphodiester following a wibonucleotide or a 3',5' -phosphodiester following an L-ribonucleotide. Both enzymes can operate in an intermolecular reaction format with multiple turnover. The DNA enzyme that cleaves a 2',5' -phosphodiester exhibits a k(sub cat) of approx. 0.01/ min and catalytic efficiency, k(sub cat)/k(sub m) of approx. 10(exp 5)/ M min. The enzyme that cleaves an L-ribonudeotide is about 10-fold slower and has a catalytic efficiency of approx. 4 x 10(exp 5)/ M min. Both enzymes require a divalent metal cation for their activity and have optimal catalytic rate at pH 7-8 and 35-50 C. In a comparison of each enzyme s activity with either its corresponding substrate that contains an unnatural ribonudeotide or a substrate that instead contains a standard ribonucleotide, the 2',5' -phosphodiester-deaving DNA enzyme exhibited a regioselectivity of 6000- fold, while the L-ribonucleotide-cleaving DNA enzyme exhibited an enantioselectivity of 50-fold. These molecules demonstrate how in vitro evolution can be used to obtain regio- and enantioselective catalysts that exhibit specificities for nonnatural analogues of biological compounds.
Document ID
20030067986
Acquisition Source
Headquarters
Document Type
Preprint (Draft being sent to journal)
Authors
Ordoukhanian, Phillip
(Scripps Research Inst. La Jolla, CA, United States)
Joyce, Gerald F.
(Scripps Research Inst. La Jolla, CA, United States)
Date Acquired
August 21, 2013
Publication Date
September 17, 2002
Subject Category
Life Sciences (General)
Funding Number(s)
CONTRACT_GRANT: SFP-1076
CONTRACT_GRANT: NAG5-4546
Distribution Limits
Public
Copyright
Other

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