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

Area of Science:

• Molecular Biology
• Biotechnology
• Diagnostics

Background:

• Precise control over nucleic acid amplification is crucial for molecular diagnostics, especially in point-of-care and field applications.
• Conventional isothermal amplification methods like recombinase-aided amplification (RAA) often lack spatiotemporal control, leading to issues like spontaneous initiation and poor synchronization.

Purpose of the Study:

• To develop light-activatable RAA systems for spatiotemporal control over nucleic acid amplification.
• To engineer photocaged components for RAA to enable on-demand activation and improve diagnostic assay performance.

Main Methods:

• Two photocaging strategies were employed: a photocaged DNA polymerase and photocaged primers.
• The photocaged polymerase involved site-specific incorporation of p-azido-L-phenylalanine and click chemistry conjugation.
• Photocaged primers were synthesized using 6-nitropiperonyloxymethyl modification to block primer function until UV activation.

Main Results:

• Both photocaging strategies effectively suppressed background amplification in the dark and restored full efficiency upon UV activation (365 nm).
• The systems demonstrated high sensitivity, low background noise, and compatibility with portable diagnostic devices.
• A custom device integrated UV activation and real-time fluorescence detection for seamless, on-demand operation.

Conclusions:

• The developed modular platform offers flexible, light-gated amplification solutions for precise molecular diagnostics.
• The choice between polymerase- or primer-based caging allows optimization for storage stability or workflow integration.
• These advancements pave the way for more reliable and field-ready molecular diagnostic tools.