Formation of synthetic RNA protein granules using engineered phage-coat-protein -RNA complexes

Abstract Liquid-solid transition, also known as gelation, is a specific form of phase separation in which the interacting molecules cross-link to form a highly interconnected compartment with solid – like dynamical properties. Here, we utilize RNA hairpin coat-protein binding sites to form synthetic RNA based gel-like granules via liquid-solid phase transition. we show both in-vitro and in-vivo that hairpin containing synthetic long non-coding RNA (slncRNA) molecules granulate into bright localized puncta. We further demonstrate that upon introduction of the coat-proteins, less-condensed gel-like granules form with the RNA creating an outer shell with the proteins mostly present inside the granule. Moreover, by tracking puncta fluorescence signals over time, we detected addition or shedding events of slncRNA-CP nucleoprotein complexes. Consequently, our granules constitute a genetically encoded storage compartment for protein and RNA with a programmable controlled release profile that is determined by the number of hairpins encoded into the RNA. Our findings have important implications for both the potential regulatory role that naturally occurring granules play and for the broader biotechnology and gene-expression sectors.