An efficient RNA-cleaving DNA enzyme that synchronizes catalysis with fluorescence signaling.
DNA enzymes are single-stranded DNA molecules with catalytic capabilities that are isolated from random-sequence DNA libraries by "in vitro selection". This new class of catalytic biomolecules has the potential of being used as unique molecular tools in a variety of innovative applications. Here we describe the creation and characterization of an RNA-cleaving autocatalytic DNA, DEC22-18, that uniquely links chemical catalysis with real-time fluorescence signaling capability in the same molecule. A trans-acting DNA molecule, DET22-18, was also developed from DEC22-18 that behaves as a true enzyme with a k(cat) of approximately 7 min(-1)-a rate constant that is the second largest ever reported for a DNA enzyme. It cleaves a chimeric RNA/DNA substrate at the lone RNA linkage surrounded by a closely spaced fluorophore-quencher pair-a unique structure that permits the synchronization of the chemical cleavage with fluorescence signaling. DET22-18 has a stem-loop structure and can be conjugated with DNA aptamers to form allosteric deoxyribozyme biosensors.
Mei
Shirley H J
SH
Department of Biochemistry, McMaster University, 1200 Main Street West, Hamilton, Ontario, L8N 3Z5 Canada.
Liu
Zhongjie
Z
Brennan
John D
JD
Li
Yingfu
Y
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D021881:DNA, Catalytic / Q000737:chemistry* / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000737:chemistry; D012313:RNA / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods; D013379:Substrate Specificity
D021881:DNA, Catalytic; D004277:DNA, Single-Stranded; D009841:Oligonucleotides; D012313:RNA; D000255:Adenosine Triphosphate