Triple-hairpin mediated feedback system based on rolling circle amplification for enhanced imaging and detection of intracellular microRNA

Abstract

It remains challenging to develop highly sensitive biosensing methods for the detection of short, non-coding RNA, such as microRNAs (miRNAs) that were expressed at low levels in biological environments. Here we report a triple-hairpin mediated feedback system based on rolling circle amplification (THF-RCA) for enhanced imaging and detection of intracellular miRNA. THF-RCA contains two RCA-based tandem reactions, Cycle I and Cycle II. Cycle I is initiated by a target-primed RCA reaction to produce the RCA product that can successively open HP1, HP2, and HP3. The target-analogue embedded in HP3 can be activated accompanied by the dissolving of HP3 and go on to initiate another RCA reaction, named Cycle II. Subsequently, the RCA product of Cycle II can initiate a new round of Cycle I in a feedback manner. Overall, the triple-hairpin structures serve as the pivot to power the THF-RCA reaction continues autonomously. THF-RCA based cycle system can be used to detect miR-21 down to 1 fM, and target miRNA can be identified obviously from mutation versions and no-target miRNAs. Notably, the THF-RCA based strategy allows not only for in vitro detection of miRNAs without reverse transcription, but also for intracellular imaging of miRNA with higher sensitivity compared with miRNA-FISH technology. THF-RCA based strategy is expected to provide new doors for developing novel signal amplification technologies and biosensors applied in early tumor diagnosis and early treatment.