Emergent Chemical Reactivity and Complexity of RNA Condensates

Abstract The RNA world hypothesis posits the existence of life-like assemblies that consisted mostly of RNA. However, questions remain regarding the emergence of RNA catalysis, stability, reactant availability, and compartmentalization of genetic material. At acidic pH, short RNAs (average ≈ 20 nt) readily phase-separate into a condensed phase en-riched with long RNA. These RNA condensates stably compartmentalize RNA as well as DNA and maintain stable identities even in the absence of membranes. Additionally, the RNA condensates concentrate ions, small molecules, phospholipids, peptides, ri-bozymes, and proteins. Beyond enriching such diverse components, RNA condensates function as microreactors with two catalytic capabilities: they physically enhance reac-tion rates by concentrating reactants within a confined space and simultaneously act as inherent catalysts that directly facilitate chemical transformations. RNA condensates can also support ribozyme and enzymatic activity. Together, these findings suggest that RNA phase separation may have played a crucial role in life’s origins by providing compartmentalization, inherent catalytic activity, and molecular enrichment of long, potentially catalytic biopolymers. Competing Interest Statement The authors have declared no competing interest. Footnotes This version of the manuscript has been revised regarding relevance, experimental rigor, and scientific depth of the presented results. Technical improvements have been made throughout the manuscript. One figure not presenting data has been removed. The other figures have been modified to include error and statistical analysis where appropriate. Figure clarity has been improved with systematic labeling. Experimental details have been added and materials descriptions improved for transparency and reproducibility. Substantial new experimental data is also provided in the main manuscript and the supplementary information. For example, data on the reversibility of RNA phase separation (Figure 2e), on RNA deamination (Figure S4), on the impact of RNA degradation by alkaline hydrolysis (Figure S5), on the liquid-like behavior of RNA condensates using FRAP (Figure S9), on amino acid transamination for the glycine-pyruvate system (Figure S13), and on direct pH measurements inside RNA condensates using LysoSensor probe (Figure S14). The quantitative studies on deamination and alkaline hydrolysis address the possibility of RNA degradation impacting the observed phenomena.