Non-canonical interactions tune condensation and kinetically arrested material states of short RNA homopolymers

Phase separation related processes have been frequently linked to biological compartmentalization and organization, and associated with cellular responses to stress, development, and disease. As a result, understanding biomolecular condensates and the parameters that modulate material and chemical properties has been an area of interest. Although much initial work focused on the role of proteins in phase separation, increasingly RNA-RNA interactions are being recognized for their potential to tune the condensate properties, influencing material properties, morphology, and dynamic organization. While many of these studies have focused on Watson-Crick base pairing as the primary networking interaction, previous work has also suggested a potential for non-canonical RNA-RNA interactions in long homopolymers to promote phase separation and modulate overall material properties. Here, we examine the phase separation of short RNA homopolymers in the presence of divalent cations and disordered peptides. Our studies reveal that particular short homopolymers form internally kinetically arrested droplets with limited monomer mixing despite continued fusion. We also probe the combined cooperative effects of various ions in modulating phase behavior and investigate the potential for homopolymer tracts in slightly more complex sequences to tune the material and phase separation propensities of different oligomers. Collectively, our results suggest a potential for tracts predominantly interacting through non-canonical interactions to tune the phase behavior and mesoscale properties of simplified condensates, as well as offer insight into the fundamental biophysics of RNA-based condensate systems and short homopolymer tracts in more complex biological systems.