Deciphering molecular mechanisms of phase separation in RNA biology by single-molecule biophysical technologies

Ribonucleic acid (RNA) biology has emerged as one of the most important areas in modern biology and biomedicine. RNA and RNA-binding proteins (RBPs) are involved in forming biomolecular condensates, which are crucial for RNA metabolism. To quantitively decipher the molecular mechanisms of RNP granules, researchers have turned to single-molecule biophysical techniques, such as single-molecule Förster resonance energy transfer (smFRET), in vivo single-molecule imaging technique with single particle tracking (SPT), DNA Curtains, optical tweezers, and atomic force microscopy (AFM). These methods are used to investigate the molecular biophysical properties within RNP granules, as well as the molecular interactions between RNA and RBPs and RBPs themselves, which are challenging to study using traditional experimental methods of the liquid-liquid phase separation (LLPS) field, such as fluorescence recovery after photobleaching (FRAP). In this work, we summarize the applications of single-molecule biophysical techniques in RNP granule studies and highlight how these methods can be used to reveal the molecular mechanisms of RNP granules.