Light-Activated Nucleic Acid Amplification Systems Using Photo-Caged DNA Polymerase or Primers.

Abstract

Precise control over nucleic acid amplification is essential for achieving reliable, quantitative, high-throughput, and multiplexed molecular diagnostics, particularly in point-of-care and field settings. However, conventional isothermal amplification methods, especially rapid assays at ambient temperature such as recombinase-aided amplification (RAA), suffer from spontaneous initiation and lack of synchronization across parallel reactions. Here, light-activatable RAA systems employing two photocaging strategies to achieve spatiotemporal control are reported. In the first, a photocaged DNA polymerase is engineered via site-specific incorporation of p-azido-L-phenylalanine and conjugation with a 2-nitrobenzyl-modified ssDNA blocker through click chemistry. This approach offers high stability, making it advantageous for long-term storage and lyophilized formats. In the second, 6-nitropiperonyloxymethyl-modified primers are synthesized to block hybridization or extension until UV activation, offering operational simplicity and rapid integration into existing workflows. Both strategies independently suppressed background activity in the dark and restored full amplification efficiency upon exposure at 365 nm. The systems exhibited high sensitivity, low background, and compatibility with portable diagnostics. A custom-built device integrating UV activation and real-time fluorescence detection enabled seamless, on-demand operation. This modular platform provides flexible light-gated amplification solutions, allowing the selection of polymerase- or primer-based caging according to storage stability or workflow requirements, thereby advancing precise and field-ready molecular diagnostics.