Characterizing protein interactions in biomolecular phase separation

Protein interactions are ubiquitous to life as they enable essential functions such as protein synthesis, signalling cascades or sequestration of foreign antigens. Pairwise interactions, in particular, have been studied extensively for centuries leading to the development of various techniques that can characterise binding events reliably. Recently, phase separation of proteins into membraneless compartments within the cellular environment has emerged as novel context of protein interactions. Although, great progress has been made in the discovery and study of phase separating systems, the protein interactions underlying these macromolecular assemblies remain difficult to characterize given their transient, multivalent nature. Here we show novel approaches for characterizing protein interactions within phase separating systems involving the usage of microfluidic tools such as microfluidic diffusional sizing and construction of tie lines from confocal detection. In doing so, we discover DNA-induced phase separation of antibodies as well as the formation of nanoscale clusters in antibody-DNA mixtures. Furthermore, we characterize the mechanistic impact of an RNA aptamer on multicomponent FUS condensate stability and determine the interactions of salt excipients with the protein dense phase. Crucially, establishing such novel pathways for characterizing protein interactions in phase separating systems will be key to accelerate the discovery of molecules that amend or interfere with phase separation, hence, show the potential to alter disease courses.