A dual-cycle DNA walker sensor for sensitive clinical detection of microRNAs

MicroRNAs (miRNAs) play crucial roles in both physiological and pathological processes, and are increasingly recognized as important biomarkers for cancers and other diseases. However, current detection methods for miRNA face challenges, including inadequate sensitivity and the need for temperature-controlled instruments, which hinder their clinical application. It is a pressing need for new strategies suitable for clinical miRNA detection.

We designed a dual-cycle DNA walker sensor (DDWS) by coupling duplex-specific nucleases enzyme-assisted signal amplification (DSNSA) with a DNA walker sensor. In the DSNSA process, the DSN enzyme specifically hydrolyzes DNA in DNA/target miRNA hybrid duplexes to facilitate the target cycling. The DNA walker sensor is activated by the trigger released from the DSNSA process, enabling a DNA walker cycle via the CHA reaction on magnetic microparticles (MMPs). The DDWS exhibited a strong linear relationship over a range of 600 fM to 600 nM and demonstrated excellent specificity for target miRNA. Furthermore, the DDWS was successfully applied to miRNA detection in real samples, including total RNAs extracted in cell samples and clinical papillary thyroid carcinoma (PTC) tissue samples.

This DDWS assay holds great potential for evaluating miRNA expression levels across various biological matrices, contributing to the clinical diagnosis and prognosis of cancers.