Specific anchoring of large topologically closed DNA for single-molecule protein:DNA interactions

Single-molecule and bulk biophysical approaches to study protein-DNA interactions on surface-immobilized nucleic acid templates typically rely on modifying the ends of linear DNA molecules to enable surface-DNA attachments. Unless both strands are constrained, this results in topologically free DNA molecules and the inability to observe supercoiling-dependent biological processes or requires additional means to micromanipulate the free DNA end to impose rotational constraints or induce supercoiling. We developed a method using RecA protein to induce the formation of a circularized compliment-stabilized D-loop. The resulting joint molecule is topologically closed, surface anchorable, and stable under microfluidic flow. Importantly, the method obviates the need for subsequent manipulation of surface-tethered DNA; tethered molecules remain supercoiled and retain accessibility to DNA-binding proteins. This approach adds to the toolkit for those studying processes on DNA that require supercoiled DNA templates or topologically constrained systems.