Biosensing by Tandem Reactions of Structure Switching, Nucleolytic Digestion, and DNA Amplification of a DNA Assembly

Angew Chem Int Ed Engl. 2015 Aug 10;54(33):9637-41. doi: 10.1002/anie.201503182. Epub 2015 Jun 26.

Abstract

ϕ29 DNA polymerase (ϕ29DP) is able to carry out repetitive rounds of DNA synthesis using a circular DNA template by rolling circle amplification (RCA). It also has the ability to execute 3'-5' digestion of single-stranded but not double-stranded DNA. A biosensor engineering strategy is presented that takes advantage of these two properties of ϕ29DP coupled with structure-switching DNA aptamers. The design employs a DNA assembly made of a circular DNA template, a DNA aptamer, and a pre-primer. The DNA assembly is unable to undergo RCA in the absence of cognate target owing to the formation of duplex structures. The presence of the target, however, triggers a structure-switching event that causes nucleolytic conversion of the pre-primer by ϕ29DP into a mature primer to facilitate RCA. This method relays target detection by the aptamer to the production of massive DNA amplicons, giving rise to dramatically enhanced detection sensitivity.

Keywords: DNA amplification; aptamers; biosensors; rolling circle amplification; structure switching.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aptamers, Nucleotide / chemistry*
  • Aptamers, Nucleotide / metabolism
  • Bacillus Phages / enzymology
  • Biosensing Techniques / methods*
  • DNA / chemistry*
  • DNA / metabolism
  • DNA, Circular / chemistry
  • DNA, Circular / metabolism
  • DNA-Directed DNA Polymerase / metabolism
  • Nucleic Acid Amplification Techniques / methods*

Substances

  • Aptamers, Nucleotide
  • DNA, Circular
  • DNA
  • DNA-Directed DNA Polymerase