Search

Rupture Event Scanning (REVS) is applied for the first time within an approach based on dynamic force spectroscopy. Using model DNA duplexes containing 20 pairs of oligonucleotides including those containing single mismatches, we demonstrated the possibility of reliable determination of the kinetic parameters of dissociation of biomolecular complexes: barrier positions, the rate constants of dissociation, and the lifetime of complexes. Within this approach, mechanical dissociation of DNA duplexes occurs according to a mechanism similar to unzipping. It is shown that this process takes place by overcoming a single energy barrier. In the case where a mismatch is located at the farthest duplex end from the QCM surface, a substantial decrease in the position of the barrier between the bound and unbound states is observed. We suppose that this is due to the formation of an initiation complex containing 3-4 pairs of bases, and this is sufficient for starting duplex unzipping.

For the first time, rupture event scanning (REVS) procedure based on quartz crystal microbalance (QCM) and involving only mechanical action was used to determine the height of the energy barrier for dsDNA unwinding. Melting point was determined with the help of this procedure. To determine the thermodynamic parameters including enthalpy, DNA denaturation was represented as a unimolecular process. This allowed us to recover the energy profiles from the experimental data obtained by force measurements at different scanning times (reaction times) for different temperatures. The thus obtained results were compared with the data obtained with the help of another mechanical method, namely, atomic force microscopy. The mechanism of DNA unwinding in QCM-based experiments through the unzipping mode, as proposed by us in previous works, was confirmed. Thus, we demonstrated that REVS procedure may be used to assess the thermodynamic parameters of dsDNA unwinding.

Rupture Event Scanning (REVS) was used to study oligonucleotide unwinding under mechanical load. Oligonucleotide melting temperature was successfully estimated using this method. To estimate the enthalpy of reaction, we represented denaturation process as a unimolecular reaction. This gave us the possibility to recover the force profile from the experimental data obtained in force measurements at different scanning time (reaction time) for different temperatures.