Ribozyme activity modulates the physical properties of RNA–peptide coacervates

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    Experimental models of simple cell-like compartments can help us to understand how biology operated early in its history. The authors convincingly show how the properties of coacervate droplets can be influenced by the activity of ribozymes inside them. This important result potentially provides a new route for biologists or chemists to establish cell mimics that support the evolution of biomolecules within.

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Condensed coacervate phases are now understood to be important features of modern cell biology, as well as valuable protocellular models in origin-of-life studies and synthetic biology. In each of these fields, the development of model systems with varied and tuneable material properties is of great importance for replicating properties of life. Here, we develop a ligase ribozyme system capable of concatenating short RNA fragments into long chains. Our results show that the formation of coacervate microdroplets with the ligase ribozyme and poly(L-lysine) enhances ribozyme rate and yield, which in turn increases the length of the anionic polymer component of the system and imparts specific physical properties to the droplets. Droplets containing active ribozyme sequences resist growth, do not wet or spread on unpassivated surfaces, and exhibit reduced transfer of RNA between droplets when compared to controls containing inactive sequences. These altered behaviours, which stem from RNA sequence and catalytic activity, constitute a specific phenotype and potential fitness advantage, opening the door to selection and evolution experiments based on a genotype–phenotype linkage.

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  1. eLife assessment

    Experimental models of simple cell-like compartments can help us to understand how biology operated early in its history. The authors convincingly show how the properties of coacervate droplets can be influenced by the activity of ribozymes inside them. This important result potentially provides a new route for biologists or chemists to establish cell mimics that support the evolution of biomolecules within.

  2. Reviewer #1 (Public Review):

    RNA-based self-replication systems might have been concentrated and compartmentalized with peptides by forming droplet-like complexes before the emergence of cellular organisms enveloped by lipid membranes. This report clearly shows that the physical properties of such droplets (phenotypes) can be affected by the activities of a ligase ribozyme in the droplets. This suggests that sequences of such ribozymes (genotypes) might have been selected not only for their direct activities (e.g., elongation of RNA) but also for their indirect effects on the droplet phenotypes (e.g., more viscous/solid droplets formed by the elongated RNAs) on the ancient earth. However, the exact requirements (e.g., average/maximum length of the ligated RNAs + double strand formation) for such phenotypic changes are not assessed in this report. It is not demonstrated whether the droplet property changes caused by the ribozyme activity can be advantageous for the survival of the RNA-based system (e.g., for the ribozyme activity itself). Follow-up studies would be desired to clarify these points and the true values of this report.

  3. Reviewer #2 (Public Review):

    To understand the origins of life, it is often necessary to establish synthetic molecular systems that model how primitive cells might have operated. Adopting this approach, here Le Vay et al. tackle one of the mysteries of early cells: how could primitive biomolecules have controlled the behavior of the compartments they inhabited? By forming coacervate droplets from polylysine peptides and ribozymes (catalytic RNAs), they observe changes in droplet properties driven by ribozyme activity and propose a route to form an integrated protocellular system that allows the evolution of biomolecules based on compartment behavior, modeling potential early life processes.

    Polymers of opposite charge can phase-separate into coacervate droplets in equilibrium with surrounding aqueous phases. Such condensates are thought to act as subcellular compartments mediating some cellular functions. Coacervates, though, are also of interest as model compartments for biomolecules at the origins of life. A number of studies have shown how the properties and behavior of coacervates can be modulated based on external biological or physicochemical changes. There remains a key question: for coacervates to serve as a vessel for biology at the origin of life, can coacervate behavior be controlled from within? Previously this has been shown possible in some systems of membranous vesicles, an alternative model of primordial compartments.

    Proteinaceous enzymes have been deployed to transform precursor compounds into potential coacervate components and induce the formation of condensed-phase microdroplets, but such enzymes are not thought to have been available at the origins of life. Instead, ribozymes are thought to have catalysed key reactions in early biology. Here, by using a ribozyme ligase to concatenate RNA molecules when together in a polylysine coacervate, the authors clearly demonstrate that coacervate properties change, showing a more rounded droplet shape and reduced fusion tendencies. Interestingly, the authors find that this distinctive behavior emerges when the reaction occurs in the coacervate phase, instead of before coacervate formation.

    This influence of sequence-encoded phenotype on compartment properties has few precedents and has long been a target of origin of life research. The authors propose that it could serve as the basis for the establishment of coacervate droplets as units of selection and evolution. For this, a trio of critical challenges must be overcome and the authors begin to shed light on these.

    First, the droplets must support ribozyme activity, without overly inhibiting it (or the droplet becomes an unfavorable habitat for these catalysts). Other ribozymes have often suffered inhibition due to conformation effects or substrate availability when mixed in a coacervate. The authors show here that the ligase ribozyme maintains activity (and may even be accelerated) in the coacervate. However, it appears to operate under single-turnover conditions and it is not yet clear whether multiple-turnover catalysis is possible in the coacervate.

    Second, the droplet properties must be responsive to the activity of the biomolecules inside. The authors' observations of changes in coacervate behavior are robust, and they make some suggestions as to how such changes might be leveraged to establish selection pressure to drive the evolution of content molecules. In this study, though, the ribozyme comprises a substantial fraction (~1/2) of the coacervate negatively charged components, and in an evolutionary situation (with fewer molecules of RNA catalyst per compartment) it is not known whether the resulting droplet phenotype will change impactfully.

    Third, the droplet must hold together its contents and avoid mixing with the contents of other droplets, to hold a molecular species together and defend against molecular parasites. Though there may still be some exchange of smaller molecules, the authors demonstrate that the lengthening of RNAs by ribozyme ligases in a coacervate can prevent fusion with other similar droplets (which otherwise occurs in the absence of RNA ligation) and preserve droplet identity. To use the coacervate as an evolutionary unit, droplets with active ribozyme will also need to be resistant to fusion with inactive droplets.

    Putting such a system together based on the phenomenon observed by the authors would be a breakthrough in modeling primordial biology. A range of compartments have been proposed to act as habitats for early molecular biology, including porous rocks, mineral surfaces, ice phases, aerosols as well as membranous vesicles, and a key challenge is demonstrating how internal biological activities can influence compartment behavior. Establishing coacervates as genetically-controllable habitats for biomolecules will add to experimental models of such "life but not as we know it" and provide a new view of early biology.

  4. Reviewer #3 (Public Review):

    The study investigates the consequences of mixing a ligase ribozyme, its substrates, and oligo(Lys) peptides of different lengths in the context of a coacervate droplet protocell in a 'Nucleic Acid World' as an early stage of life. The study shows convincingly several very interesting results that are certain to have an impact on origins-of-life studies: First, the activity of ribozymes in the coacervate droplets - the formation of longer RNAs - affects the size of the droplets, with inactive ribozymes leading to more droplet fusion. Second, this behavior is reflected in the adhesion to hydrophobic surfaces, showing that not only the size but also the physical properties of the droplets are changed by ribozyme catalysis. Third, the exchange rate of material between droplets is also affected by ribozyme catalysis, which has important implications for coacervates as model systems for early life forms.

    More detailed information should be provided in the text that ribozyme catalysis actually proceeds in/on the coacervates, a discussion section needs to be devoted to the implication of ribozyme catalysis affecting the measured material exchange rates on the coupling of genotype/phenotype, molecular parasites, and the inflow/outflow of metabolites, and the importance of the system with longer peptides needs to be clarified and perhaps toned down.