A light-up "G-quadruplex nanostring" for label-free and selective detection of miRNA via duplex-specific nuclease mediated tandem rolling circle amplification

Abstract

MicroRNA (miRNA) has emerged as a promising genetic marker for cancer diagnosis and therapy because its expression level is closely related to the progression of malignant diseases. Herein, a label-free and selective fluorescence platform was proposed for miRNA based on light-up "G-quadruplex nanostring" via duplex-specific nuclease (DSN) mediated tandem rolling circle amplification (RCA). First, a long DNA generated from upstream RCA was designed with the antisense sequences for miR-21 and downstream RCA primer. Upon recognizing miR-21, the resulting DNA-RNA permitted DSN digestion and triggered downstream two-way RCA, and generation of abundant "G-quadruplex nanostring" binding with ZnPPIX for label-free fluorescent responses. In our strategy, the strong preference of DSN for perfectly matched DNA/RNA ensures its excellent selectivity. The developed method generated wide linear response with LOD of 1.019 fM. Additionally, the miR-21 levels in cell extracts have been evaluated, revealing the utility of this tool for biomedical research and clinical diagnosis.

Keywords: Duplex-specific nuclease; G-quadruplex nanostring; MicroRNA; Tandem rolling circle amplification.