title: A novel molecular beacon-based method for isothermal detection of sequence-specific DNA via T7 RNA polymerase-aided target regeneration.
abstract: Developing molecular beacon (MB)-based method for DNA detection has been of great interest to many researchers because of its intrinsic advantages of simplicity, rapidity, and specificity. In this work, we have developed a novel MB-based method for isothermal detection of sequence-specific DNA via T7 RNA polymerase-aided target regeneration strategy. The proposed method involves three primary processes of target-mediated ligation by T4 DNA ligase, transcription reaction by T7 RNA polymerase, and MB switch for signal output. Upon the hybridization with DNA target, a rationally designed MB and a pair of primers encoded with T7 promoter sequence were ligated via the formation of a phosphodiester bond by T4 DNA ligase. The resultant joint fragment acted as template to initiate T7 RNA polymerase-mediated transcription reaction. Correspondingly, a great amount of RNA strands complementary to MB and partial primers were transcribed to initiate new cyclic reactions of MB switch, ligation, and transcription. With such signal amplification strategy of the regeneration of target-like RNA fragments, our proposed assay achieved a detection limit as low as ∼10 pM, which was ∼3 orders of magnitude lower than the traditional MB-based method with a recognition mechanism in 1:1 stoichiometric ratio between MB and target molecule.
authors.affiliation: Lab of Biosystem and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China University of Science & Technology, Shanghai 200237, China.
authors.forename: Bin-Cheng
authors.initials: BC
authors.lastname: Yin
authors.affiliation: Lab of Biosystem and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China University of Science & Technology, Shanghai 200237, China.
authors.forename: Shan
authors.initials: S
authors.lastname: Wu
authors.affiliation: Lab of Biosystem and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China University of Science & Technology, Shanghai 200237, China.
authors.forename: Jin-Liang
authors.initials: JL
authors.lastname: Ma
authors.affiliation: Lab of Biosystem and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China University of Science & Technology, Shanghai 200237, China. Electronic address: bcye@ecust.edu.cn.
authors.forename: Bang-Ce
authors.initials: BC
authors.lastname: Ye
mesh_terms: D017123:Bacteriophage T7 / Q000201:enzymology*; D001483:Base Sequence; D015374:Biosensing Techniques / Q000379:methods*; D004247:DNA / Q000032:analysis* / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D057230:Limit of Detection; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D009693:Nucleic Acid Hybridization / Q000379:methods*; D014764:Viral Proteins / Q000378:metabolism*
chemical_list: D014764:Viral Proteins; D004247:DNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases
keywords: Isothermal detction; Molecular beacon; Nucleic acid detection; Signal amplification