title: In vitro selection of RNA-cleaving DNAzymes for bacterial detection.
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.
authors.affiliation: Department of Biochemistry and Biological Sciences, McMaster University, Hamilton, Ont. L8S 4K1, Canada.
authors.forename: Wenqing
authors.initials: W
authors.lastname: Zhang
authors.affiliation: Department of Chemistry and Chemical Biology, McMaster University, Hamilton, Ont. L8S 4K1, Canada.
authors.forename: Qian
authors.initials: Q
authors.lastname: Feng
authors.affiliation: Department of Biochemistry and Biological Sciences, McMaster University, Hamilton, Ont. L8S 4K1, Canada.
authors.forename: Dingran
authors.initials: D
authors.lastname: Chang
authors.affiliation: Department of Biochemistry and Biological Sciences, McMaster University, Hamilton, Ont. L8S 4K1, Canada.
authors.forename: Kha
authors.initials: K
authors.lastname: Tram
authors.affiliation: Department of Biochemistry and Biological Sciences, McMaster University, Hamilton, Ont. L8S 4K1, Canada. Electronic address: liying@mcmaster.ca.
authors.forename: Yingfu
authors.initials: Y
authors.lastname: Li
mesh_terms: D001419:Bacteria / Q000235:genetics / Q000302:isolation & purification; D015374:Biosensing Techniques / Q000379:methods*; D021881:DNA, Catalytic / Q000737:chemistry / Q000235:genetics*; D004277:DNA, Single-Stranded; D015202:Protein Engineering / Q000379:methods*; D012313:RNA / Q000737:chemistry / Q000235:genetics*
chemical_list: D021881:DNA, Catalytic; D004277:DNA, Single-Stranded; D012313:RNA
keywords: Bacterial detection; Biosensor; Clostridium difficile; DNAzyme; In vitro selection; RNA cleavage