FRAP4ICBS: FRAP Analysis Method to Evaluate Dominant Effects for Biomolecular Condensate Formation.

Cells contain a wide variety of membrane-less organelles, whose formation mechanism is believed to be generally based on liquid-liquid phase separation (LLPS). In fact, condensate formation can involve more than one mechanism; interactions with spatially clustered binding sites (ICBS) may contribute alongside LLPS, sometimes dominating and sometimes playing only a partial role. Here, we introduce FRAP4ICBS, a FRAP analysis method based on ICBS mechanism to characterize the dominant formation mechanisms of condensates. FRAP4ICBS can accurately distinguish between the formation mechanisms of condensates as either LLPS- or ICBS-dominant both in vitro and in silico. We also found that condensates that require DNA/RNA involvement are more likely to be based on the ICBS-dominant mechanism. On this basis, we tested the FUS-ERG protein and DNA co-condensates, which was often claimed to be based on LLPS, and demonstrated that it is more likely to be produced by the ICBS-dominant mechanism. In addition, this conclusion is also verified by single particle tracking. Therefore, the FRAP4ICBS can serve as a tool for identifying the potential mechanisms underlying condensate formation, offering support for a deeper understanding of the formation mechanism of condensates.