Can one detect intermediate denaturation states of DNA sequences by following the equilibrium open-close dynamic fluctuations of a single base-pair?*

Melting of DNA sequences may occur through a few major intermediate states, whose influence has been suggested previously on the melting curve, while their effect on the kinetics has not been explored thoroughly. Here we chose a simple DNA sequence, forming a hairpin in its native (zipped) state, and study it using molecular dynamic (MD) simulations, and a model integrating the Gaussian network model with bond-binding energies -- the Gaussian binding energy (GBE) model. We reveal two major partial denaturation states, a bubble state, and a partial unzipping state. We show how these two states can influence the closing-opening base-pair dynamics as probed by a tagged bond auto-correlation function (ACF). We argue that the latter is measured by fluorescence correlation microscopy (FCS) experiments in which one base of the pair is linked to a fluorescent dye while the complementary base is linked to a quencher, as was performed by Altan-Bonnet et al. [Phys. Rev. Lett. 90, 138101 (2003)]. We find that tagging certain base pairs at temperatures around the melting temperature results in a multi-step relaxation of the ACF, while tagging other base pairs leads to an effectively single-step relaxation, albeit non-exponential. Only the latter type of relaxation has been observed experimentally so far, and we suggest which of the other base pairs should be tagged in order to observe multi-step relaxation. We demonstrate that this behavior can be observed with other sequences, and argue that the GBE can reliably predict these dynamics for very long sequences, including those used to construct DNA origami, where MD simulations are very limited by computer resources.