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RNA-cleaving DNA enzymes with altered regio- or enantioselectivity

Academic Article
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Overview

authors

  • Ordoukhanian, Phillip
  • Joyce, Gerald

publication date

  • 2002

journal

  • Journal of the American Chemical Society  Journal

abstract

  • In vitro evolution methods were used to obtain DNA enzymes that cleave either a 2',5'-phosphodiester following a D-ribonucleotide 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(cat) of approximately 0.01 min(-1) and catalytic efficiency, k(cat)/K(m), of approximately 10(8) M(-1) min(-1). The enzyme that cleaves an L-ribonucleotide is about 10-fold slower and has a catalytic efficiency of approximately 4 x 10(5) M(-1) min(-1). Both enzymes require a divalent metal cation for their activity and have optimal catalytic rate at pH 7-8 and 35-50 degrees C. In a comparison of each enzyme's activity with either its corresponding substrate that contains an unnatural ribonucleotide or a substrate that instead contains a standard ribonucleotide, the 2',5'-phosphodiester-cleaving DNA enzyme exhibited a regioselectivity of 6000-fold, while the L-ribonucleotide-cleaving DNA enzyme exhibited an enantioselectivity of 40-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.

subject areas

  • DNA, Catalytic
  • Kinetics
  • Nucleic Acid Conformation
  • Oligonucleotides
  • RNA
  • Stereoisomerism
  • Substrate Specificity
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Identity

International Standard Serial Number (ISSN)

  • 0002-7863

Digital Object Identifier (DOI)

  • 10.1021/ja027467p

PubMed ID

  • 12381192
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Additional Document Info

start page

  • 12499

end page

  • 12506

volume

  • 124

issue

  • 42

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