Single-Molecule Approaches to Study DNA Condensation.

Abstract

Proteins drive genome compartmentalization across different length scales. While the identities of these proteins have been well-studied, the physical mechanisms that drive genome organization have remained largely elusive. Studying these mechanisms is challenging owing to a lack of methodologies to parametrize physical models in cellular contexts. Furthermore, because of the complex, entangled, and dense nature of chromatin, conventional live imaging approaches often lack the spatial resolution to dissect these principles. In this chapter, we will describe how to image the interactions of λ-DNA with proteins under purified and cytoplasmic conditions. First, we will outline how to prepare biotinylated DNA, functionalize coverslips with biotin-conjugatedpoly-ethylene glycol (PEG), and assemble DNA microchannels compatible for the imaging of protein-DNA interactions using total internal fluorescence microscopy. Then we will describe experimental methods to image protein-DNA interactions in vitro and DNAloop extrusion using Xenopus laevis egg extracts.