3 years ago

Specific and Direct Amplified Detection of MicroRNA with MicroRNA:Argonaute-2 Cleavage (miRACle) Beacons

Specific and Direct Amplified Detection of MicroRNA with MicroRNA:Argonaute-2 Cleavage (miRACle) Beacons
Benjamin M. Luby, Gang Zheng
MicroRNA detection is a valuable method for determining cell identity. Molecular beacons are elegant sensors that can transform intracellular microRNA concentration into a fluorescence intensity. While target binding enhances beacon fluorescence, the degree of enhancement is insufficient for demanding applications. The addition of specialty nucleases can enable target recycling and signal amplification, but this process complicates the assay. We have developed and characterized a class of beacons that are susceptible to the endogenous nuclease Argonaute-2 (Ago2). After purification of the complex by co-immunoprecipitation, microRNA:Ago2 cleavage (miRACle) beacons undergo site- and sequence-specific cleavage, and show a 13-fold fluorescence enhancement over traditional beacons. The system can be adapted to any microRNA sequence, and can cleave nuclease-resistant, non-RNA bases, potentially allowing miRACle beacons to be designed for cells without interference from non-specific nucleases. Simple sensors simulate substrate: MicroRNA detection with molecular beacons can be used for in situ genetic profiling. Beacons that are susceptible to the endogenous nuclease Argonaute-2 (Ago2) have now been developed. After purification of the complex by co-immunoprecipitation, microRNA:Ago2 cleavage (miRACle) beacons undergo site- and sequence-specific cleavage, and show a 13-fold fluorescence enhancement over traditional beacons.

Publisher URL: http://onlinelibrary.wiley.com/resolve/doi

DOI: 10.1002/anie.201707366

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