In Vitro Characterization of Protein:Nucleic Acid Liquid–Liquid Phase Separation by Microscopy Methods and Nanoparticle Tracking Analysis

AbstractUncontrolled assembly/disassembly of physiologically formed liquid condensates is linked to irreversible aggregation. Hence, the quest for understanding protein-misfolding disease mechanism might lie in the studies of protein:nucleic acid coacervation. Several proteins with intrinsically disordered regions as well as nucleic acids undergo phase separation in the cellular context, and this process is key to physiological signaling and is related to pathologies. Phase separation is reproducible in vitro by mixing the target recombinant protein with specific nucleic acids at various stoichiometric ratios and then examined by microscopy and nanotracking methods presented herein. We describe protocols to qualitatively assess hallmarks of protein-rich condensates, characterize their structure using intrinsic and extrinsic dyes, quantify them, and analyze their morphology over time. Analysis by nanoparticle tracking provides information on the concentration and diameter of high-order protein oligomers formed in the presence of nucleic acid. Using the model protein (globular domain of recombinant murine PrP) and DNA aptamers (high-affinity oligonucleotides with 25 nucleotides in length), we provide examples of a systematic screening of liquid–liquid phase separation in vitro.Key wordsBiomolecular condensatesLiquid-liquid phase separation (LLPS)CoacervationPhase transitionsPrion protein (PrP)DNA aptamersDifferential interference contrast (DIC) microscopyFluorescence microscopyTransmission electron microscopy (TEM)Nanoparticle tracking analysis (NTA)Amyloid fibrils