Ultrasensitive assay based on a combined cascade amplification by nicking-mediated rolling circle amplification and symmetric strand-displacement amplification.

ABSTRACT

MicroRNAs (miRNAs) play an important role in the regulation of various biological processes and have been used as potential biomarkers for biomedical research and clinical diagnosis. Here, nicking-mediated rolling circle amplification (N-RCA)/symmetric isothermal circular strand-displacement amplification (S-SDA)-integrated combined cascade amplification (rs-CCA) was proposed for let-7a miRNA detection. Via introducing a palindromic fragment-integrated recognition sites for nicking endonuclease Nt.AlWI into the padlock probe, the hybridization of target miRNA can induce N-RCA and continuously generate the nicked fragments (NFs). Because the released NFs each have a palindromic sequence and daughter nicking site at the 3' end, they hybridize with each other, followed by alternately extension by polymerase and nicking by endonuclease. This leads to S-SDA effect, and the released single-stranded products in turn hybridize with NFs, accomplishing the rs-CCA process. When the Sybr Green I dyes intercalate into double-stranded DNA products, the amplified fluorescence signal is achieved. Thus, the target miRNA can be detected down to 5 pM. Importantly, the rs-CCA system is capable of distinguishing the single base difference between target miRNAs, indicating the high sequence specificity. Moreover, its potential application in disease diagnosis was demonstrated via detecting target miRNA in complex biological matrix and analyzing the total RNAs extracted from HeLa cells. As a proof-of-concept building, the impressive rs-CCA scheme is expected to provide a valuable insight into constructing powerful signal amplification strategies via sophisticated combination of biotechnologies available for nucleic acid manipulation and significantly benefit biomedical research and disease diagnostics.