Genetic encoding and mutagenesis of RNA droplet phenotypes.

RNA droplets assembled from cotranscriptionally folded nanostructures have recently emerged as a promising platform for constructing protocells and minimal synthetic cell models. A custom-designed DNA template encodes an RNA nanostar that self-assembles into droplets by kissing-loop interactions. Here we show that such RNA droplets encapsulate their DNA template, creating a genotype-phenotype link. We find that low mutation rates in the DNA template increase the phenotypical and functional complexity of the RNA droplets. Surprisingly, mutagenesis leads to the formation of long-lived vacuoles. With theory and next-generation sequencing, we explain this emergent behavior. We identify mutant nanostars that facilitate the formation of vacuoles through altered interactions between nanostars. Strikingly, vacuolated droplets exhibit micrometer-scale movement driven by internal vacuole dynamics - an emergent function resulting from mutagenesis. Beyond motility, we show that the vacuoles can be used as subcompartments to store cargo in the RNA droplets. Our findings establish RNA droplets as self-contained, evolvable systems, opening new avenues for the bottom-up evolution of synthetic cells and origins-of-life research.