Twinkle-Catalyzed Toehold-Mediated DNA Strand Displacement Reaction

Strand exchange between homologous nucleic acid sequences is the basis for cellular DNA repair, recombination, and genome editing technologies. Specialized enzymes catalyze cellular strand exchange; however, the reaction occurs spontaneously when a single-stranded DNA toehold can dock the invader strand on the target DNA to initiate strand exchange through branch migration. Due to its precise response, the spontaneous toehold-mediated strand displacement (TMSD) reaction is widely employed in DNA nanotechnology. However, enzyme-free TMSD suffers from slow rates, resulting in slow response times. Here, we show that human mitochondrial DNA helicase Twinkle can accelerate TMSD up to 6000-fold. Mechanistic studies indicate that Twinkle accelerates TMSD by catalyzing the docking step, which typically limits spontaneous reactions. The catalysis occurs without ATP, and Twinkle-catalyzed TMSD rates remain sensitive to base-pair mismatches. The simple catalysis, tunability, and speed improvement of the catalyzed TMSD can be leveraged in nanotechnology, requiring sensitive detection and faster response times.