An efficient RNA-cleaving DNA enzyme that synchronizes catalysis with fluorescence signaling

J Am Chem Soc. 2003 Jan 15;125(2):412-20. doi: 10.1021/ja0281232.

Abstract

DNA enzymes are single-stranded DNA molecules with catalytic capabilities that are isolated from random-sequence DNA libraries by "in vitro selection". This new class of catalytic biomolecules has the potential of being used as unique molecular tools in a variety of innovative applications. Here we describe the creation and characterization of an RNA-cleaving autocatalytic DNA, DEC22-18, that uniquely links chemical catalysis with real-time fluorescence signaling capability in the same molecule. A trans-acting DNA molecule, DET22-18, was also developed from DEC22-18 that behaves as a true enzyme with a k(cat) of approximately 7 min(-1)-a rate constant that is the second largest ever reported for a DNA enzyme. It cleaves a chimeric RNA/DNA substrate at the lone RNA linkage surrounded by a closely spaced fluorophore-quencher pair-a unique structure that permits the synchronization of the chemical cleavage with fluorescence signaling. DET22-18 has a stem-loop structure and can be conjugated with DNA aptamers to form allosteric deoxyribozyme biosensors.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / chemistry
  • Adenosine Triphosphate / metabolism
  • Base Sequence
  • Catalysis
  • Cloning, Molecular
  • DNA, Catalytic / chemistry*
  • DNA, Catalytic / metabolism
  • DNA, Single-Stranded / chemistry*
  • DNA, Single-Stranded / metabolism
  • Kinetics
  • Molecular Sequence Data
  • Nucleic Acid Conformation
  • Oligonucleotides / chemical synthesis
  • Oligonucleotides / chemistry
  • RNA / chemistry*
  • RNA / metabolism
  • Spectrometry, Fluorescence / methods
  • Substrate Specificity

Substances

  • DNA, Catalytic
  • DNA, Single-Stranded
  • Oligonucleotides
  • RNA
  • Adenosine Triphosphate