Mitochondrial microRNA detection using a sequentially activatable allosteric DNA biosensor for in vivo molecular...

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.