Direct observation of effect of crowding induced macromolecular hydration on molecular breathing in the stem of Fork-DNA by single-molecule FRET microspectroscopy

The perpetually changing cellular conditions, nucleotide sequence, and environmental effects including osmotic stress have multiple effects on DNA, leading to several conformational alternations and subsequently influencing their activity, too. In this work, single-molecule FRET microspectroscopy has been employed to monitor the breathing dynamics as an effect of molecular crowding in the stem region of Fork-DNA. The structural integrity greatly alters with the presence or absence of nucleotide overhangs and on the nature and concentration of the crowding agent, thus affecting the stability of the stem region and hence the forked DNA. The multiple hydrogen bonds and hydrophobic interactions between the polynucleotide strands appear to be altered with osmotic crowding. This induces increased flexibility in the double helix and allows DNA to breath. The conformational alternation of the DNA happens in nanometer resolution, that is been monitored by the change in the FRET efficiency between the dyes attached to two different strands of the DNA. The nature and molecular weight of crowding agents control the degree of spatial breathing in the stem of Fork-DNA. These constant fluctuations between the entropically favorable partially folded structures to an enthalpically favorable folded structure are not only valuable for elucidating nucleic acid structure but might play an important role in enzyme kinetics. (C) 2020 Elsevier B.V. All rights reserved.