Protein recruitment to dynamic DNA-RNA host condensates

Living cells through biomolecular condensates and their phase separation can spatiotemporally control and localize small molecules and proteins. Taking inspiration from nature, we propose and characterize synthetic DNA-RNA condensates that are capable of spatially localizing and recruiting proteins of interest. We design programmable subunits, termed nanostars and made up of three DNA strands and one RNA strand, that have valency three and phase separate into a dense phase. We engineer an aptamer domain on the RNA strand for the recruitment of a target protein; hence, the engineered nanostars will function as 'host' molecules. As a model protein we consider Streptavidin (SA) because of its widespread use in binding assays. In addition to demonstrating SA recruitment, we describe two mechanisms to control the onset of condensation and recruitment. The first mechanism employs UV irradiation as a physical stimulus that bypasses the need for exchanging molecular inputs, thus is particularly convenient to control and investigate condensation in confinement, as we illustrate through encapsulation of condensates in water in oil droplets. The second mechanism benefits from RNA transcription, a biochemical reaction that is key to the development of living soft materials. We show that the combination of RNA transcription and RNA degradation leads to an autonomous dissipative system in which host condensates and protein recruitment occur transiently. In our system, condensate size as well as the timescale of the transient can be controlled by the level of RNA degrading enzyme. Lastly, we demonstrate that biotinylated beads can be recruited to SA-host condensates successfully and with minimal entropic cost, suggesting a robust phase separation of the host nanostars which may therefore find practical application for the spatial separation of a variety of biotin-tagged components.