GLUTARALDEHYDE BASED IN-VIVO SNAP-CROSSLINKING AMENABLE TO BIOCONDENSATE SEPARATION FOR COMPOSITIONAL IDENTITY BASED ANALYSIS

Membrane-bound organelles organize their molecular events by regulated transport of molecules through their transmembrane channels. On the other hand, MLOs are non-stoichiometric assemblies of locally concentrated molecules mainly proteins, RNAs and DNAs. MLOs are condensed liquid-like structures that appear distinct from their milieu. These condensates are highly dynamic and quickly exchange their components with their surrounding environment, resulting in a vast number of transient interactions. In this work, we focused on developing a robust protocol that is amenable for different biocondensates as applied to Cytoplasmic G3BP1 and Nuclear FUS which are major components of Stress Granules and Paraspeckles respectively. In our study, we compared various chemical cross-linking agents that can rapidly crosslink transient and higher-order complexes unlike canonical protocols where fixation is allowed for longer time points or that limit crosslinking to pairwise interactions. Further, the major challenge of extraction from such higher-order-crosslinked complexes either cytosolic or nuclear localized is dealt with a novel lysis protocol which can be used for specific immunocapture and marker-lncRNA analysis of the captured complexes.