Protein Recruitment\nto Dynamic DNA-RNA Host Condensates

We describe the design and characterization of artificial\nnucleic\nacid condensates that are engineered to recruit and locally concentrate\nproteins of interest <i>in vitro</i>. These condensates\nemerge from the programmed interactions of nanostructured motifs assembling\nfrom three DNA strands and one RNA strand that can include an aptamer\ndomain for the recruitment of a target protein. Because condensates\nare designed to form regardless of the presence of target protein,\nthey function as “host” compartments. As a model protein,\nwe consider Streptavidin (SA) due to its widespread use in binding\nassays. In addition to demonstrating protein recruitment, we describe\ntwo approaches to control the onset of condensation and protein recruitment.\nThe first approach uses UV irradiation, a physical stimulus that bypasses\nthe need for exchanging molecular inputs and is particularly convenient\nto control condensation in emulsion droplets. The second approach\nuses RNA transcription, a ubiquitous biochemical reaction that is\ncentral to the development of the next generation of living materials.\nWe then show that the combination of RNA transcription and degradation\nleads to an autonomous dissipative system in which host condensates\nand protein recruitment occur transiently and that the host condensate\nsize as well as the time scale of the transition can be controlled\nby the level of RNA-degrading enzyme. We conclude by demonstrating\nthat biotinylated beads can be recruited to SA-host condensates, which\nmay therefore find immediate use for the physical separation of a\nvariety of biotin-tagged components.