Elucidating the Role of Topological Constraint on the Structure of Overstretched DNA Using Fluorescence Polarization Microscopy

The combination of DNA force spectroscopy and polarization microscopy fluorescent intercalator dyes can provide valuable insights into the structure under tension. These techniques have previously been used to characterize S-DNA—an elongated conformation that forms when overstretches at forces ? 65 pN. In this way, it was deduced base pairs S-DNA are highly inclined, relative those in relaxed (B-form) DNA. However, is unclear whether how topological constraints on may influence base-pair inclinations Here, we apply investigate impact pulling geometry, torsional constraint, negative supercoiling orientations intercalated during overstretching. contrast earlier predictions, geometry (namely, molecule stretched via opposite strands or same strand) found little influence. constraint leads a substantial reduction tilting overstretched DNA, particularly AT-rich sequences. Surprisingly, extent similarly reduced negatively supercoiled up critical density (corresponding ?70% linking number). We attribute these observations presence P-DNA (an overwound conformation). Our results suggest preferentially flanks regions rather than also substantiate previous suggestions predominantly