# Universal Regulation of DNA Polymerase via Photocaged Primer Enables Light‐Start Isothermal Amplification on Demand

## TL;DR

A new method uses light to control DNA copying enzymes, improving the accuracy and timing of DNA amplification for diagnostics.

## Contribution

A universal photocaged primer approach is introduced to enable light-start isothermal amplification with enhanced specificity and temporal control.

## Key findings

- The photocaged primer inhibits DNA polymerase conformational changes until activated by UV light.
- Light-start RPA improves reaction specificity and integrates well into digital droplet workflows.
- Light-start RPA detects pathogens with 100% sensitivity and specificity within 20 minutes.

## Abstract

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.

A simple and general photoactivatable approach is developed to regulate DNA polymerase activity, enabling light‐start isothermal amplification on demand. The molecular dynamics simulations reveals that the photocaged primer blocks DNA polymerase activity by inhibiting the conformational change of the polymerase. Moreover, the proposed light‐start recombinase polymerase amplification (Light‐start RPA) improves reaction specificity and provides superior temporal control.