A mismatch-suppressed, duplex-specific nuclease powered nanowalker for multiplexed sensing of microRNA.

Abstract DNA molecular machines have attracted immense interest for their potential in biosensing, drug delivery, and cellular imaging. Herein, we report a duplex-specific nuclease (DSN) powered nanowalker that can autonomously and progressively move on a spherical three-dimensional track, which is constructed by functionalizing a 13 nm diameter gold nanoparticle (AuNP) with densely mismatched DNA duplexes. The motion is initiated by an RNA walking strand, and in its absence, the walker is suppressed because the DSN is inactive toward the mismatched DNA duplexes. Once the walking strand is added, perfectly matched DNA-RNA hybrid is formed via a toehold-mediated displacement reaction between the walking strand and mismatched duplex. Thereafter, the DNA-RNA hybrid is simultaneously cleaved by DSN, by releasing the walking strand, which autonomously moves on the track with the aid of DSN. The present study provides a novel energy input and power mechanism for the operation of 3-D nanowalker with high efficiency. Moreover, the proposed nanowalker can be designed in a target microRNA (miRNA)-specific manner by altering the mismatched duplexes, and it exhibits femtomole level sensitivity in both singleplexed and multiplexed sensing of three miRNA targets. In addition, multiplexed quantification of the three miRNAs in biological samples is achieved, further suggesting that the proposed nanowalker has immense potential in biomedical research and early diagnosis of clinical disorders.