Molecular crowding suppresses mechanical stress-driven DNA strand separation.

Molecular crowding influences DNA mechanics and DNA-protein interactions and is ubiquitous in living cells. Quantifying the effects of molecular crowding on DNA supercoiling is essential to relating in vitro experiments to in vivo DNA supercoiling. We use single-molecule magnetic tweezers to study DNA supercoiling in the presence of dehydrating or crowding cosolutes. To study DNA supercoiling, we apply a stretching force of 0.8 pN to the DNA and then rotate one end of the DNA to induce supercoiling. In a 200 mM NaCl buffer without cosolutes, negatively supercoiled DNA absorbs some of the tortional stress by forming locally melted DNA regions. The base pairs in these locally melted regions are believed to adopt a configuration where nucleotide base pairing is disrupted. We find that the presence of a dehydrating cosolute such as glycerol further destabilizes base pairs in negatively supercoiled DNA. The presence of polyethylene glycol, commonly used as a crowding agent, suppresses local strand separation and results in plectoneme formation even when DNA is negatively supercoiled. The results presented in this letter suggest further directions for studies of DNA supercoiling and supercoiled DNA-protein interactions in molecular conditions that approximate in vivo molecular composition.