Probing Multiple Binding Modes of DNA Hybridization: A Comparison between Single-Molecule Observations and Ensemble Measurements

[Image: see text] Interactions between biomolecules are generally analyzed by ensemble measurements, assuming that the interactions occur in a single binding manner. However, such interactions may occur via multiple binding modes. We investigated the kinetics of DNA hybridization as a multiple dynamic model of biomolecular interactions. Two kinetic analyses were performed with a single-molecule observation using total internal reflection fluorescence microscopy (TIRFM) and with ensemble measurements using a quartz-crystal microbalance (QCM) biosensor. We observed the DNA hybridization of 8 and 12 bp DNAs with random sequences and dA(12)–dT(12) and calculated the kinetic parameters, including the dissociation rate constant (k(off)). Hybridization of 8 bp DNA proceeded mainly via a single binding mode. However, hybridization of 12 bp DNA indicated at least two different binding modes and dA(12)–dT(12) hybridization showed multiple binding modes. For the multiple binding interactions, the kinetic parameters obtained from TIRFM and QCM were different because kinetic parameters obtained from QCM indicate average number of molecules, whereas those from TIRFM indicate average association time. The present study revealed the details of multiple interactions, which can be utilized for better understanding of not only DNA hybridization but also biomolecular interaction mechanisms.