Imaging compaction of single supercoiled DNA molecules by atomic force microscopy

Abstract

Supercoiled pGEMEX DNA, 3993 bp in length, was immobilized on different substrates (freshly cleaved mica, standard amino mica and modified amino mica with increased hydrophobicity and surface charge density compared with standard amino mica) and was visualized by atomic force microscopy (AFM) in air. Plectonemically supercoiled DNA (scDNA) molecules, as well as extremely compacted single molecules, were visualized on amino-modified mica, characterized by increased hydrophobicity and surface charge density. We show four-fold increase in DNA folding on the mica surface with high positive charge density. This result is consistent with a strongly enhanced molecular flexibility facilitated by shielding of the DNA phosphate charges. The formation of minitoroids with about a 50 nm diameter and molecules in spherical conformation was the final stage of single molecule compaction. A possible model of conformational transitions for scDNA in vitro in the absence of protein is proposed based on AFM image analysis. Compaction of the single scDNA molecules, up to minitoroids and spheroids, appears to be caused by screening of the negatively charged DNA phosphate groups. The high surface charge density from positively charged amino groups on mica, on which DNA molecules were immobilized, is an obvious candidate for the screening effect.