Enhancing Amplification Efficiency and Reducing Molecular Diagnostic Reaction Time Through Rational Design of T4 gp32 Variants in Recombinase Polymerase Amplification

Recombinase polymerase amplification (RPA) is a prominent isothermal nucleic acid amplification method widely applied in molecular diagnostics. The stability and functionality of the single-stranded DNA-binding protein T4 gene 32 (gp32) crucial for pre-synaptic filament formation and D-loop stabilization, play a key role in determining RPA efficiency. In this study, V62C/T80C and Y186R mutants with improved performance were screened by rational disulfide bond construction and virtual saturation mutagenesis, respectively. The structural changes in V62C/T80C and the altered ssDNA-binding capacity in Y186R both contribute to RPA amplification by enhancing the formation of UvsX-ssDNA presynaptic filaments and stabilizing the D-loop structure during homologous recombination, respectively. The two mutants each demonstrated unique advantages in the RPA process. V62C/T80C significantly accelerates the amplification process, reducing the RPA reaction time by 47%, while Y186R showed a 123% increase in efficiency across the entire amplification cycle. Totally, this study applied a rational strategy on gp32 optimization, shortening RPA reaction times, enhancing the RPA reaction efficiency, and advancing its application in clinical and point-of-care diagnostics.