Measuring And Modeling The Effect Of Single Mismatch On Dna Strand Displacement

DNA strand displacement is an essential reaction in both DNA homologous recombination and DNA mismatch repair, and it is also heavily utilized in DNA-based computation and locomotion. Despite this ubiquity in science and engineering, sequence-dependent effects of displacement kinetics have not been extensively characterized. Furthermore, the bulk methods currently used make it difficult to study the underlying branch migration process. Here, we directly measured the branch migration kinetics of toehold-mediated strand displacement using single-molecule fluorescence in the presence of a single nucleotide mismatch. The apparent displacement rate varied significantly when the mismatch was introduced in the invading DNA strand. The rate generally decreased as the mismatch in the invader was encountered earlier in displacement. Our data reveal direct dissociation of the shorter DNA strand (incumbent) due to weakened base pairing during branch migration as the dominant pathway for DNA strand displacement. We show that a simple first passage model can quantitatively explain the salient features of the observed relationship. We expect our model to become a powerful tool to design DNA-based reaction schemes with broad functionality.