Fluorescence detection of DNA by the catalytic activation of an aptamer/thrombin complex

J Am Chem Soc. 2005 May 11;127(18):6522-3. doi: 10.1021/ja050678k.

Abstract

A conjugate consisting of a thrombin aptamer tethered to the thrombin, Th, with a sensing nucleic acid (1) is used for the optical detection of DNA. The thrombin/aptamer complex blocks the biocatalytic functions of Th. Hybridization of the analyte DNA (2) to the sensing nucleic acid 1 yields a rigid duplex that detaches the aptamer from Th, a process that activates the protein toward the hydrolysis of bis(p-tosyl-Gly-Pro-Arg)-R110 (3) to the rhodamine 110 fluorophore (4). The system allows the DNA sensing with a sensitivity limit of 1 x 10-8 M. The aptamer/Th conjugate is also immobilized on glass slides for the optical detection of DNA. The dissociation of the aptamer/Th complex upon hybridization and the subsequent dehybridization of the duplex and the regeneration of the catalytically inactive Th/aptamer complex duplicate machinery functions.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Catalysis
  • DNA / analysis*
  • DNA / chemistry
  • DNA, Single-Stranded / analysis
  • DNA, Single-Stranded / chemistry
  • Enzyme Activation
  • Fluorescent Dyes / chemistry*
  • Models, Molecular
  • Oligonucleotides / chemistry*
  • Rhodamines / chemistry
  • Spectrometry, Fluorescence / methods*
  • Thrombin / chemistry*
  • Thrombin / metabolism

Substances

  • DNA, Single-Stranded
  • Fluorescent Dyes
  • Oligonucleotides
  • Rhodamines
  • DNA
  • Thrombin