Ribozyme-phenotype coupling in peptide-based coacervate protocells

Abstract

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 extremely long chains. Our results show that 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.

Phase-separated droplets can direct the kinetics of chemical reactions including polymerization, self-replication and oscillating networks

“…However, for larger 𝐾 𝑃 (𝐾 𝑃 > 30), the reaction in the dense phase starts to determine the overall reactions (Supplementary Figure 15), as under these conditions the majority of the reactant mass is in the dense phase and the majority of T is formed there. By inhibiting transfer between the phases for individual components (Supplementary Figure 16- 22), we were able to find that transfer of ABT from the dense to the dilute phase is critical to obtain the contributions in Figure 3c. Formation of ABT is the most unfavorable step, as it is the step with the highest molecularity.…”

“…The functioning of self-replicators inside coacervate protocells has recently received a lot of attention. Several self-replicating DNA and ribozyme systems have been shown to function inside coacervates, 22,25,62 and in some cases the replication rate is even enhanced. 13 How well self-replication works is highly dependent on the nature of the coacervate material.…”

“…[8][9][10][11][12][13][14][15][16] Additionally, the local polarity, crowding, pH and water activity can have substantial effects on the energy landscape of reactions, affecting both reaction rates and pathways. 17 A wide range of reactions have been shown to be enhanced by localization to a coacervate, including enzymatic 8,12,[14][15][16]18 and nanoparticle-catalysed 8,19 reactions, ribozyme reactions, 13,[20][21][22] reactions between synthetic and prebiotically-relevant small molecules, [9][10][11]23,24 template-directed RNA polymerization, 25 DNA ligation 26 and cell-free gene expression. 27 Our group has recently also shown that coacervates can lead to preferential formation of specific products in peptide ligation by oxidative coupling of α-amidothioacids and amino acids, 24 showcasing that coacervates can not only accelerate reactions, but also direct reaction pathways.…”

Nucleic acid liquids

“…For example, Schoenmakers et al created in vitro biomolecular coacervates with specific compositions of proteins and NAs capable of performing both transcription and translation [96]. Le Vay et al found that, within droplets formed from coacervation of ribozymes (an RNA enzyme) and PLL, the ribozymes displayed an enhanced catalytic rate and yield [97], and further that droplets containing active ribozymes displayed different physical properties (e.g. in droplet growth and adhesion) than those containing inactive ribozymes.…”

How Droplets Can Accelerate Reactions─Coacervate Protocells as Catalytic Microcompartments

“…Uptake of the ribozyme and its substrate(s) can be achieved by partitioning into preformed coacervates, 17,19,22,23 or by condensation of the ribozyme and substrate with cationic peptides into RNA-based coacervates (Figure 4.b). 20,21,50 The selected compartmentalization strategy has implications for the local concentration of ribozymes and their substrates: partitioning yields a local concentration of HH-ribozyme of 50 μM (K p = 9600), 23 whereas condensation is estimated to result in local oligonucleotide concentrations as high as several mM. 48,50 However, despite the increased local concentration, not all studies reported a rate enhancement inside coacervates.…”