In vitro selection of RNA-cleaving DNAzymes for bacterial detection

Methods. 2016 Aug 15;106:66-75. doi: 10.1016/j.ymeth.2016.03.018. Epub 2016 Mar 24.

Abstract

DNAzymes refer to single-stranded DNA molecules with catalytic activity and can be isolated from synthetic random-sequence DNA pools using the technique of in vitro selection. DNAzymes that cleave RNA, known as "RNA-cleaving DNAzymes", represent one of the best-studied classes of DNAzymes and have been widely used for the development of biosensors and bioassays for various analytes. We have been interested in developing RNA-cleaving DNAzymes as bacterial sensors and these DNAzymes are engineered to perform three linked functions: recognition of a bacterial biomarker, RNA cleavage, and fluorescence generation. These fluorogenic DNAzymes emit fluorescence automatically in the presence of a bacterium of interest and can be used to set up a simple "mix-and-read" assay to detect this bacterium. In this article, we will discuss this DNAzyme system and present a proven strategy for isolating highly specific bacteria-responding DNAzyme probes from random-sequence DNA pools. We will also provide an in vitro selection protocol successfully used to derive RNA-cleaving fluorogenic DNAzyme probes that are capable of recognizing a targeted strain of Clostridium difficile.

Keywords: Bacterial detection; Biosensor; Clostridium difficile; DNAzyme; In vitro selection; RNA cleavage.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacteria / genetics
  • Bacteria / isolation & purification
  • Biosensing Techniques / methods*
  • DNA, Catalytic / chemistry
  • DNA, Catalytic / genetics*
  • DNA, Single-Stranded
  • Protein Engineering / methods*
  • RNA / chemistry
  • RNA / genetics*

Substances

  • DNA, Catalytic
  • DNA, Single-Stranded
  • RNA

Grant support