Enzyme translocation-mediated domino-type DNA nanomachine for in vivo specific imaging of mitochondrial ATP

The abnormal localization of mitochondrial adenosine triphosphate (ATP) is closely linked to tumor proliferation, migration, and drug resistance. While ATP is widely distributed in cells, such as in the cytoplasm, nucleus, organelles, and cell membranes. Consequently, the development of strategies with precise localization control for in situ imaging of mitochondrial ATP poses a significant challenge. Therefore, an apurinic/apyrimidinic endonuclease 1 (APE1) translocation-mediated domino-type DNA nanomachine (C-ATP-tFNA) was developed, which can target mitochondria by using the mitochondria-specific localization of cytochrome c (cyt c) as the targeting agent. The interaction between S5-hairpin 1 (S5-HP1) of C-ATP-tFNA and cyt c induces a conformational change in S5-HP1 to HP1, thereby exposing the AP site for cleavage by mitochondrial APE1 and releasing a single-strand cyclic DNA sequence (cyclic sequence), which leads to the first configurational change of C-ATP-tFNA. Then, the cyclic sequence can be hybridized with hairpin 2 (HP2) of C-ATP-tFNA in a circular mode, leading to a second cleavage by APE1 in a domino effect and thus causing the second configuration change of C-ATP-tFNA. Finally, upon cleavage of the AP site on HP2 by APE1, the ATP aptamer on HP2 is activated and gains the ability to bind ATP, thus generating a fluorescence signal in the presence of ATP. The experimental results of this study demonstrated that C-ATP-tFNA can achieve highly specificity and sensitivity in vivo imaging of mitochondrial ATP in tumor and inflammatory cells. In particular, C-ATP-tFNA can monitor drug resistance in neuroblastoma (NB) in vivo , offering a novel and reliable approach for the surveillance of NB drug resistance.