Single-molecule and ensemble methods to probe RNP nucleation and condensate properties.

Biomolecular condensates often consist of intrinsically disordered protein and RNA molecules, which together promote the formation of membraneless organelles in cells. The nucleation, condensation, and maturation of condensates is a critical yet poorly understood process. Here, we present single-molecule and accompanying ensemble methods to quantify these processes more comprehensively. In particular, we focus on how to properly design and execute a single-molecule nucleation assay, in which we detect signals arising from individual units of fluorescently labeled RNA-binding proteins associating with an RNA substrate. The analysis of this data allows one to determine the kinetics involved with each step of nucleation. Complemented with meso-scale techniques that measure the biophysical properties of ribonucleoprotein condensates, the methods described herein are powerful tools that can be adopted for studying any protein-RNA interactions that promote phase separation.