Generalizable 3′-Hydroxyl-Regenerating Primer Platform for Photoactivatable DNA Polymerase Regulation in Nucleic Acid Amplification

Abstract

Precise regulation of DNA polymerase activity remains a central challenge in nucleic acid amplification, particularly in systems susceptible to nonspecific amplification. Here, we report a generalizable 3′-hydroxyl-regenerating primer platform for photoactivatable DNA polymerase regulation in nucleic acid amplification. This platform exploits the conserved requirement of polymerase function, namely the recognition of a free 3′-hydroxyl terminus for both primer extension and exonuclease initiation. By engineering a nonhydroxylated 3′ terminus linked to a photocleavable moiety, both processes are suppressed prior to activation. Upon light exposure, restoration of the native 3′-hydroxyl group triggers initiation of DNA synthesis, enabling precise temporal control of polymerase activity. Mechanistic studies reveal that the nonhydroxyl 3′ terminus inhibits DNA polymerase activity by preventing the catalytically required conformational transition. This approach eliminates sequence-dependent design constraints and enables broad compatibility across diverse amplification systems. The platform is applied to both isothermal and thermocycling formats, enabling photoactivatable recombinase polymerase amplification and quantitative PCR with robust performance. Photoactivatable qPCR enabled H1N1 plasmid detection down to 10 ag/μL, whereas photoactivatable recombinase polymerase amplification detected EMP1 targets down to 100 ag/μL. It supports RNA detection and multiplexed analysis while effectively suppressing premature amplification. Clinical validation further demonstrates rapid and accurate detection in real samples, achieving complete agreement with an NMPA-approved RT-qPCR method within 20 min. Overall, this work establishes a general framework for 3′-terminus engineering-based control of DNA polymerase activity, enabling temporal regulation of nucleic acid amplification across diverse applications.