Ultrasensitive Fluorescence Detection and Imaging of MicroRNA in Cells Based on a Hyperbranched RCA-Assisted Multiposition SDR Signal Amplification Strategy

Herein, an innovative fluorescent sensor was courageously empoldered for precise and ultrasensitive detection and imaging of target miRNA-21 through the agency of a dextrous target-motivated polymerization/nicking DNA nanomachineries based on a hyperbranched rolling circle amplification (HB-RCA)-assisted multiposition strand displacement reaction (SDR) signal amplification approach. Impressively, the ingenious technique not only realized target recycling via polymerization/nicking DNA nanomachineries but also involved HB-RCA amplification induced by the released transformation target as the repeated signal amplification. Most importantly, HB-RCA was firstly exploited to remarkably increase the local concentration and collision efficiency of the templates and primers, which could simultaneously generate multiple repeated DNA sequences as initiators to supply substantial banding positions for SDR, removing the massive fluorescence-resonance-energy-transfer (FRET) DNA duplexes from the repeated DNA sequences to remarkably avert the self-quenching of the fluorescence signal due to self-aggregation caused by the winding of the HB-RCA products, thereby leading to a conspicuously improved signal amplification multiplier. As proof of concept, an ingenious technique effectively and accurately distinguished target miRNA-21 even with a tiny change in cells compared to the conventional fluorescence in situ hybridization (FISH) approach. Moreover, the proposed fluorescent method apparently discriminated drug-manipulative miRNA expression level abnormities. Therefore, the proposed cascade nucleic acid amplification strategy could provide an epigamic avenue for ultrasensitive imaging of diverse biomarkers, which help researchers to better study the tumor mechanism, thereby unambiguously increasing cancer cure rates and reducing the risk of recurrence.

Automated summary

In this study, an innovative fluorescent sensor was developed for precise and ultrasensitive detection and imaging of target miRNA-21. The sensor utilized a dextrous target-motivated polymerization/nicking DNA nanomachineries and a hyperbranched rolling circle amplification (HB-RCA)-assisted multiposition strand displacement reaction (SDR) signal amplification approach. The technique achieved target recycling through polymerization/nicking DNA nanomachineries and HB-RCA amplification induced by the released transformation target. HB-RCA was exploited to increase the local concentration and collision efficiency of the templates and primers, generating multiple repeated DNA sequences as initiators for SDR, which improved the signal amplification multiplier. The technique effectively distinguished target miRNA-21 and detected drug-manipulative miRNA expression level abnormities. The proposed cascade nucleic acid amplification strategy could offer a promising pathway for ultrasensitive imaging of diverse biomarkers.