A programmable and site-specific anchoring system for biophysical analysis of DNA:protein interactions on large topologically closed DNA molecules

Abstract

ABSTRACTSingle-molecule and bulk biophysical approaches to study protein-DNA interactions on surface-immobilised 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 micro-manipulate the free DNA end to impose rotational constraints or induce supercoiling. We developed a method using RecA protein to induce the formation of a circularised compliment-stabilised 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.WHY IT MATTERSSupercoiling plays an important role in regulating genetic processes such as DNA replication and transcription. We have developed a facile method to immobilise large (supra diffraction-limited) supercoiled DNA substrates without the need for complex trapping modalities to allow biophysical interrogation of the surface-tethered DNA molecules. This will expand the toolkit for experimentalists interested in studying protein-DNA interactions at the single-molecule or ensemble level requiring the use of topologically closed or supercoiling-dependent systems such as origin-dependent bacterial DNA replication.