Mitochondrial microRNA detection using a sequentially activatable allosteric DNA biosensor for in vivo molecular visualization of tumors and tumor drug resistance

Abstract

The development of strategies that enable in situ detection of mitochondrial microRNA (miRNA) remains a significant challenge, as miRNA is widely distributed across various cellular compartments. Therefore, by utilizing the mitochondria-specific localization of cytochrome c (cyt c) as the targeting moiety, a sequentially activatable allosteric DNA biosensor (C-M-tFNA) was developed for the in situ detection of mitochondrial miRNA. In the first configurational change of C-M-tFNA, the interaction between cyt c aptamer-hairpin 1 (apt-HP1) of C-M-tFNA and cyt c triggers a conformational transition in apt-HP1 to HP1, thereby exposing the apurinic/apyrimidinic site (AP site) for cleavage by mitochondrial apurinic/apyrimidinic endonuclease 1 (APE1) and releasing a single-strand cyclic DNA (cyclic sequence). In the second configurational change of C-M-tFNA, the cyclic sequence can hybridize with the green loop on hairpin 2 (HP2) of C-M-tFNA in a circular manner, resulting in a second cleavage by APE1. Finally, in the third configurational change of C-M-tFNA, miRNA can specifically hybridize with the red loop of HP2, inducing a third cleavage mediated by APE1. This process effectively separates the fluorophore from the quencher in a circular manner, leading to the generation of fluorescence signal. Experimental results demonstrate that C-M-tFNA enables highly specific and sensitive in vivo imaging of mitochondrial miRNA. In particular, C-M-tFNA is capable of monitoring drug resistance in neuroblastoma in vivo.