Fig. 1: APE1 detects target and generates DNA guide for PfAgo.
From: NAPTUNE: nucleic acids and protein biomarkers testing via ultra-sensitive nucleases escalation
a Detailed view of the PfAgo-gDNA-tDNA structure, illustrating how PfAgo bypasses the first base of the guide DNA. b The diagram depicts PfAgo’s interaction with guide RNA in the presence of magnesium, requiring a phosphate group. c Schematic diagram of APE1 for cleavage of AP site. Blue strand showing target and designed strand with AP site showing red. [Created in BioRender. Dbs, D. (2025) https://BioRender.com/j94m460]. d Detailed hypothesized mechanism where APE1-generated DNA fragments with a 5′ phosphate are loaded into PfAgo as guide DNA. This panel shows APE1 cleavage, guide DNA loading, and targeting by PfAgo in a DNA-guided manner. e Denaturing PAGE (polyacrylamide gel electrophoresis) displaying the APE1 activity for DNA-RNA structure. This experiment was repeated three times. f Denaturing PAGE showing the PfAgo nuclease activity for cleavage of P2 within the context of APE1 digestion reaction. This experiment was repeated three times. g Schematic depiction of dephosphorylation of 5′ terminal labeled phosphate group by ALP unable to trigger PfAgo activity. [Created in BioRender. Dbs, D. (2025) https://BioRender.com/j94m460]. h Denaturing PAGE showing the inactivity of PfAgo under the introduction ALP into the APE1 digestion reaction. This experiment was repeated three times. i Real-time profiles of signal response using fluorophore-quencher labeled P2 enable monitoring of the cleavage of P2 by PfAgo-gDNA. All the experiments were conducted in triplicate and graphs were represented by mean (bold line) ± standard deviation (SD).
