Kineto-Mechanistic Investigation of Effect of Macromolecular Crowding on the Breathing of DNA Bubble.

Abstract

Macromolecular crowding along with hydrogen bonding or stacking interactions and hydration reportedly has enormous repercussions on elementary biochemical processes, such as the folding of proteins or nucleic acids involving the stability of DNA base pairing. By using the mismatch-induced DNA bubble as a mesoscopic model, the complex interplay of macromolecular crowding on the dynamical fluctuations at the bubble region within the thermodynamic limit has been monitored using single-molecule fluorescence resonance energy transfer (sm-FRET). These single-molecule experimental results have been further corroborated using physical models such as "scaled particle theory" (SPT) and "Gaussian cloud model" (GCM), to predict the biological activity of DNA. The two-state fluctuation of the DNA bubble has been visualized as a function of the nature, size, and concentration of the crowder. The influence of crowders on the DNA conformation has been investigated with the help of the m-factor, the eccentricity, and the kinetic and thermodynamic parameters without any prior assumption. The clear effect of crowding on the dynamics of such a simple biomolecular system emphasizes the power of single-molecule methods and the dependency of the radius of gyration of the co-solute as well as the preferential interaction with the crowder on the distinct conformational states adopted by the bubble. This study provides an idea and hypothesizes the preferential propensity of the DNA bubble to adopt a conformation with the single-stranded domains being far apart, independent of the crowder size, that may be beneficial for efficient recognition by proteins for an uninterrupted procession of the biological process of the central dogma.