Nicking site enzyme assisted catalytic hairpin assembly based scaffold for sensitive monitoring of miRNA-21

As a class of non-coding RNA nucleotides, miRNAs has been widely regarded as an effective clinical biomarker for diagnosis and therapy of a variety of disease, such as colorectal cancer. However, development of an easy-to-operate and high-sensitive miRNA detection methods remains a huge challenge due to the low expression level, and high similarity with homologous RNA nucleotides. Herein, a novel CHA (catalytic hairpin assembly)-based sensing platform was established for sensitive, efficient and reliable in vitro detection of miRNA-21. The CHA based scaffold was constructed in a one-step self-assembly process of designed DNA probes (a S strand, a pair of hairpin probes). The presence of target miRNA initiates CHA process to form double-strand DNA (dsDNA) complex between the pair of hairpin probes, exposing nicking enzyme recognizing site. Nb.BbvCI identifies recognizing site in dsDNA section and generates a nick. With DNA polymerase based chain extension and replacement, liberated chain attends a next CHA process and a signal circle is formed. Compared with CHA, the method significant improves the sensitivity for miRNA-21 detection in vitro, showing a wider application for trace sample detection.