Spatial Control of Cleavage Site Accessibility via 12S rRNA Gating on Tetrahedral Nucleic Acid Scaffold Enables Mitochondrial APE1 Imaging for In Vivo Neuroblastoma Risk Stratification

Abstract Mitochondrial apurinic/apyrimidinic endonuclease 1 (APE1) mislocalization is closely linked to tumor progression and prognosis. Yet, its ubiquitous distribution across mitochondria, cytoplasm, and nucleus makes specific in situ imaging of mitochondrial APE1 highly challenging. Herein, we fabricate a sequentially activated biosensor (C-12S-tFNA) with 12S rRNA-gated spatial regulation of APE1 cleavage site (apurinic/apyrimidinic site, AP site) accessibility, where cytochrome c (cyt c) acts as the targeting motif to drive precise mitochondrial enrichment. In the initial state, the AP site is structurally locked to prevent nonspecific cleavage by extramitochondrial APE1. After entering the mitochondria, mitochondrial 12S rRNA, which is consistently expressed across diverse cell types, hybridizes with the AP-site-containing strand via base complementarity, leading to AP site unlocking. The exposed AP site is subsequently cleaved by mitochondrial APE1, separating the fluorophore-quencher pair and generating a fluorescent signal. Benefiting from the 12S rRNA-gated design, false-positive signals can be effectively suppressed. Experimental findings demonstrate that C-12S-tFNA exhibits high sensitivity (LOD: 0.002 U/mL) and exceptional specificity for mitochondrial APE1. Notably, it distinguishes high-risk from low-risk neuroblastoma (NB) in vivo without the need for surgically resected or biopsied tissue, while exhibiting favorable biocompatibility. Collectively, C-12S-tFNA enables noninvasive, activity-based mitochondrial APE1 imaging and holds promise for improving precision risk stratification in NB.