Genetic encoding and mutagenesis of RNA droplet phenotypes

RNA droplets assembled from co-transcriptionally folded nanostructures have recently emerged as a promising platform for constructing protocells and minimal synthetic cell models. In these systems, a custom-designed DNA template encodes an RNA nanostar, which is produced by transcription and self-assembles into droplets by kissing-loop interactions. Here, we show that such RNA droplets encapsulate their own DNA template, creating a genotype-phenotype link as the key prerequisite for evolution. We find that low mutation rates in the DNA template increase the phenotypical and functional complexity of the RNA droplets - enabling stable vacuole formation. With reverse-engineering and next-generation sequencing, we identify specific nanostar architectures that act as surfactants and are responsible for this morphological transition. Strikingly, vacuolated droplets exhibit micrometer-scale movement driven by internal vacuole dynamics, leading to enhanced fusion and growth - an emergent functional phenotype that results from mutagenesis. 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.