Double Self‐Amplified Programmable Allosteric DNA Nanomachine for Enzymatically Triggered Spatially Controllable Molecular Imaging

Abstract In vivo optical tumor molecular imaging encounters significant challenges in achieving adequate tumor specificity and sensitivity, largely attributed to off‐tumor signal leakage and the relatively low expression levels of target molecules. Therefore, a double self‐amplified programmable allosteric DNA nanomachine (named HPs‐tFNA) is developed through two elaborately designed hairpin structures (HP1 and HP2) hybridized on tetrahedral framework DNA (tFNA), enabling rapid, specific, and sensitive tumor molecular imaging using the highly specific expression of apurinic/apyrimidinic endonuclease 1 (APE1) in the tumor cytoplasm as a stimulus‐response target. In the presence of APE1, HP2 modifies two apurinic/apyrimidinic sites (AP sites), which can be specifically recognized and cleaved by APE1, releasing a significant number of cyclic sequences (cyclic‐seq) and achieving initial APE1‐assisted signal amplification. Subsequently, cyclic‐seq hybridizes with HP1, inducing a conformational change that converts the stem‐loop structure of HP1 to a linear form. This structural change facilitates the spatial separation of the fluorophore and quencher, thereby generating fluorescence signals. Furthermore, APE1 incises two AP sites within the HP1 loop region, resulting in the release of cyclic‐seq. The released cyclic‐seq can hybridize with additional HP1 to continuously amplify the fluorescence signal in a cyclic manner, thereby achieving the second round of signal amplification assisted by APE1. The experimental results of this study demonstrated that HPs‐tFNA can achieve rapid in situ tumor molecular imaging and guide precise surgical excision in vivo, with superior spatial specificity. In particular, HPs‐tFNA can effectively monitor drug resistance in neuroblastoma cells and stratify risk levels of neuroblastoma via plasma analysis.