Sub‐Ensemble Monitoring of DNA Strand Displacement Using Multiparameter Single‐Molecule FRET

Abstract

Non-enzymatic DNA strand displacement is an important mechanism in dynamic DNA nanotechnology. Here, we show that the large parameter space that is accessible by single-molecule FRET is ideal for the simultaneous monitoring of multiple reactants and products of DNA strand exchange reactions. We monitored the strand displacement from double-stranded DNA (dsDNA) by single-stranded DNA (ssDNA) at 37 °C; the data were modelled as a second-order reaction approaching equilibrium, with a rate constant of 10 m s . We also followed the displacement from a DNA three-way junction (3WJ) by ssDNA. The presence of three internal mismatched bases in the middle of the invading strand did not prevent displacement from the 3WJ, but reduced the second-order rate constant by about 50 %. We attribute strand exchange in the dsDNA and 3WJ to a zero-toehold pathway from the blunt-ended duplex arms. The single-molecule approach demonstrated here will be useful for studying complex DNA networks.

Heterogeneous migration routes of DNA triplet repeat slip-outs

“…( 57 ) Indeed, a single strand of DNA with a run of three mismatched bases was capable of displacing an otherwise complementary strand from a 3WJ, albeit at a reduced rate. ( 58 ) Therefore, mismatches per se are not a major obstacle. We have also presented evidence that the 3WJ dynamics increase with repeat length.…”