DNA tetrahedron-radiated nanonetwork for the dual-mode detection of FEN1 and T4 PNK activities

The sensitive and accurate detection of FEN1 and T4 PNK activities is of vital significance for the early diagnosis and therapeutic monitoring of cancer. Herein, we constructed a DNA tetrahedron-radiated nanonetwork (DTRN) based on multi-stage rolling circle amplifications (RCAs) for sensitive and dual-mode detection of FEN1 and T4 PNK activities. Specifically, the design included a DNA tetrahedron nanoprobe with four first-stage trigger sequences for first-stage RCAs, recognition elements and circular substrates serving as RCA templates, as well as a hairpin probe with multi-stage trigger sequence for multi-stage RCAs. Initially, FEN1 or T4 PNK-specific recognition events initiated ligation reactions, which induced the first-stage trigger sequences to activate first-stage RCAs, synthesizing long single-stranded DNAs with repetitive sequences. By recognizing these repetitive sequences, the hairpin probes initiated multi-site second-stage RCAs and further activated multi-stage RCAs. Eventually, numerous multi-stage trigger sequences extended simultaneously from different sites, generating a nanonetwork centered on the DNA tetrahedron, while concomitantly producing fluorescent and UV-vis absorption signals for quantification. The DTRN system achieved exponential enhancement in amplification efficiency through multi-stage RCAs on the DNA tetrahedron, improving detection sensitivity. The detection limit were as low as 4.58 × 10^-3 U mL^-1 of FEN1 and 5.76 × 10^-4 U mL^-1 of T4 PNK in fluorescent mode, 2.45 × 10^-2 U mL^-1 of FEN1 and 3.75 × 10^-3 U mL^-1 of T4 PNK in colorimetric mode. Moreover, the dual-mode detection facilitated mutual verification and complementation, enhancing detection accuracy. Furthermore, the successful detection of dual enzyme activities in cellular extracts confirmed that the DTRN system is a highly potent detection tool.