Biomolecular condensates have emerged as an important organizational element in cells, encoding physical properties with numerous functional links. Growing evidence has demonstrated both heterogeneity within condensates as well as potential structural and functional changes at condensate interfaces. Hence, there is an emerging need for further understanding the interplay between species’ organization, conformation, and dynamics at a single-molecule level. However, direct measurements relating structure, dynamics, and spatial positioning within droplets at a single-molecule level can be challenging due to the difficulty of monitoring dynamic and structural behavior in complex, high density chemical environments at high resolution. Here, we describe the development of a novel approach, using single molecule fluorescence-based techniques to probe diffusion dynamics and biomolecular conformation within condensates by combining FRET with MINFLUX. We demonstrate an ability to resolve different FRET constructs in model complex coacervates using this combined MINFLUX-FRET experiment, acquiring spatially- and temporally resolved FRET data by tracking single molecules. Our new, single-molecule approach has the potential to collect diffusion, spatial, and conformational data in a single experiment, which could have powerful implications for deeper understanding of structure and function of complex condensate systems, including for IDP-based condensates and in-cell studies.
Tom et al. (Sun,) studied this question.