Fig. 2: NAPTUNE development based on in situ cascade.

a Schematic for the working mechanism of NAPTUNE for recognition of nucleic acids. Briefly, probe 1 binds to RNA target and APE1 cuts the AP site of DNA-RNA structure, leading to target cycling for amplified signal output. Meanwhile, the APE1-cleaved product can be regarded as a gDNA for PfAgo system, resulting in two consecutive cutting with the additional P2 and P3, which leads to resulting in gradual signal enhancement by in situ cascade circuit. [Created in BioRender. Dbs, D. (2025) https://BioRender.com/j94m460]. b Denaturing PAGE showing the PfAgo nuclease activity for cleavage of P1, P2, and P3 within the context of APE1 digestion reaction. This experiment was repeated three times. c Real-time monitoring of signal changes due to cleavage on FAM-BHQ1-labeled probes. The reaction begins with APE1-P1, followed by the addition of PfAgo-P2 at 45 min and P3 at 70 min, respectively. d Real-time analysis of pre-mixed APE1-PfAgo cascade signal amplification, the reaction of the control group is conducted only with probes and without target RNA, APE1 in the reaction are overlaid for comparison, all the probes are labeled with FAM-BHQ1 fluorophore. e Quantification and comparison of the sensitivity of APE1-P1, APE1-P1-PfAgo-P2 (NAPTUNE without P3), and APE1-P1-PfAgo-P2-P3 (NAPTUNE) demonstrate that NAPTUNE offers a 1000-fold improvement in detection sensitivity compared to using APE1 alone. NC, negative control assay with buffer to substitute miR-21 target. Each group consists of 12 parallel assays in a microplate reader (Tecan Spark GmbH, Austria). Statistical analysis was conducted using a two-tailed t-test. Statistical significance was determined as follows: ns (not significant) for p > 0.05, * for p ≤ 0.05, ** for p ≤ 0.01, *** for p ≤ 0.001, and **** for p ≤ 0.0001.