Developing fluorogenic RNA-cleaving DNAzymes for biosensing applications.
Deoxyribozymes (or DNAzymes) are single-stranded DNA molecules that have the ability to catalyze a chemical reaction. Currently, DNAzymes have to be isolated from random-sequence DNA libraries by a process known as in vitro selection (IVS) because no naturally occurring DNAzyme has been discovered. Several IVS studies have led to the isolation of many RNA-cleaving DNAzymes (RNase DNAzymes), which catalyze the transesterification of a phosphodiester linkage in an RNA substrate, resulting in its cleavage. An RNase DNAzyme and its substrate can be modified with a pair of donor and acceptor fluorophores (or a fluorophore and quencher pair) to create a fluorescence-signaling system (a signaling DNAzyme) where the RNA-cleaving activity of the DNAzyme is reported through the generation of a fluorescent signal. A signaling DNAzyme can be further coupled with an aptamer (a target-binding nucleic acid sequence) to generate a fluorogenic aptazyme in which the aptamer-target interaction confers an allosteric control of the coupled RNA-cleaving and fluorescence-signaling activity of the DNAzyme. Fluorogenic aptazymes can be exploited as valuable molecular tools for biosensing applications. In this chapter, we provide both a detailed description of methods for isolation of signaling DNAzymes by IVS and general approaches for rational engineering of fluorogenic aptazymes for target detection.
Department of Biochemistry, McMaster University, Hamilton, ON, Canada.
M Monsur
MM
Ali
Sergio D
SD
Aguirre
Wendy W K
WW
Mok
Yingfu
Y
Li
D052157:Aptamers, Nucleotide / Q000378:metabolism; D001483:Base Sequence; D015374:Biosensing Techniques / Q000379:methods*; D003001:Cloning, Molecular; D021881:DNA, Catalytic / Q000235:genetics / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism*; D015723:Gene Library; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012313:RNA / Q000378:metabolism*; D017421:Sequence Analysis
D052157:Aptamers, Nucleotide; D021881:DNA, Catalytic; D005456:Fluorescent Dyes; D012313:RNA