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.