High-fidelity telomerase activity assay based on light-triggered nucleic acid separation system for the diagnosis of bladder cancer.

In conventional methods for telomerase activity assay, the obtained telomerase sample contains a large amount of impurity, which seriously affect the accuracy of the assay. Herein, we propose a light-triggered nucleic acid separation strategy to realize high fidelity Telomerase activity assay (Lit-Telo). In the light-triggered nucleic acid separation system, a 5' terminal biotinylated and photo-cleavable (PC) linker-functionalized telomerase substrate probe (Bio-PCTS) is designed. Telomerase extends telomeric repeat DNA (TTAGGG) to the 3' terminal of Bio-PCTS probe to produce telomerase extension product, which can be captured by streptavidin coated 96-well plate. Thus, the impurity can be removed from the reaction to realize the purification of telomerase extension product. A few seconds of UV light irradiation can disrupt the PC-linker in Bio-PCTS probe, allowing the easy and quick release of the telomerase extension product DNA fragment from the bottom of 96-well plate into the reaction solution for subsequent detection. Asymmetric-PCR-based TRAP and LbaCas12a/crRNA system were elucidated and optimized to realize the enhanced detection of telomerase activity. The proposed Lit-Telo platform achieved a limit-of-detection of telomerase activity equivalent to 8 HeLa cells. 26 bladder specimens were collected for telomerase activity assay using both fluorescence detection based Lit-Telo (Fluo Lit-Telo) visual detection based Lit-Telo (Visual Lit-Telo). ROC (receiver operating characteristic curve) analysis of the data indicated the good detection accuracy of Fluo Lit-Telo and Visual Lit-Telo methods with the AUC value of 93.94% and 92.12%, respectively. These results demonstrated the potential of the Lit-Telo platform in the in vitro diagnosis of bladder cancer.