Universal Regulation of DNA Polymerase via Photocaged Primer Enables Light-Start Isothermal Amplification on Demand.

The development of DNA polymerases with controllable functions, such as hot-start or light-start variants, allows on-demand activation of enzymatic activity and therefore improves reaction specificity. However, the fine control of DNA polymerase activity faces challenges to accuracy and flexibility. Here, a simple and general photoactivatable approach for the temporal regulation of DNA polymerase activity, and thus, light-start isothermal amplification on demand is engineered. This approach leverages photocaged primer with light-sensitive 6-nitropiperonyloxymethyl to temporarily inhibit the conformational change of DNA polymerase until reactivation by 365 nm UV. The mechanism through biochemical assays and molecular dynamic simulations is elucidated, and validate the temporal precision using droplet digital technology. The light-start recombinase polymerase amplification (Light-start RPA) and demostrates that it improves reaction specificity and provides superior temporal control, thereby integrating better into digital droplet workflows is further developed. Moreover, Light-start RPA offers sensitivity, specificity, and multiplexing capability for pathogen detection. Notably, both RT-Light-start RPA and Light-start RPA-based lateral flow assays demonstrate 100% sensitivity and specificity for influenza A virus detection within 20 min. Overall, this photocaged primer approach not only expands the conditional control toolbox of DNA polymerase activity, but also provides a versatile and promising framework for molecular diagnostics and microfluidics.