DNA curtains for studying phase separation mechanisms of DNA-organizing proteins.

Abstract

Phase separation is one key mechanism to organize chromatin into compartments and to regulate the activity of the genome. The formation of liquid-like droplets within the nucleus is driven by protein association to the DNA via multivalent binding and the recruitment of other proteins building a concentrated reaction environment. Common methods to study phase separation and its liquid-like nature are based on microscopy of the formed droplets but lack the resolution to obtain information on the molecular level. Here, we describe the application of the DNA curtain technique for studying protein-mediated phase separation on DNA. For this, multiple lipid-anchored DNA strands are flow-stretched across a nanobarrier to allow single-molecule studies of protein-DNA interactions in a high-throughput approach. Our protocol describes how protein-induced DNA compaction can be observed in real-time and which wash protocols are suitable to characterize the interactions that promote condensate formation. Furthermore, we demonstrate how fluorescently labeled tracer proteins can serve as orientation points to examine the DNA compaction mechanism in detail.