Many biological biomolecular condensates are formed from RNA and proteins or peptides (Roden and Gladfelter, 2021; Ukmar-Godec et al., 2019), and coacervate phases formed by charge-mediated phase separation are part of the mechanism that drives the development of membraneless organelles in modern biology (Hyman et al., 2014; Wang et al., 2021). Condensed phases studied in the context of origin of life can also be formed from RNA and peptides. Catalytic nucleic acids, including ribozymes and DNAzymes, are central to ‘Nucleic Acid World’ hypotheses, where they act as both the medium of information storage and the catalyst for its replication in early life-like systems. Peptide–RNA condensation forms discrete protocellular compartments (Abbas et al., 2021; Oparin, 1965). RNA–peptide interactions are in many cases beneficial for the function of these early catalysts (Frenkel-Pinter et al., 2020; Tagami et al., 2017), and may promote the folding and oligomerisation of certain peptides (Seal et al., 2022). Perhaps surprisingly, the function of ribozymes and other nucleic acid enzymes in coacervate phases has only been recently established, with catalytic rate being enhanced and enzyme function altered in some cases (Drobot et al., 2018; Poudyal et al., 2019a; ; Poudyal et al., 2019b; Iglesias-Artola et al., 2022). For example, coacervation with poly(L-lysine) shifts the reaction equilibrium of a minimal hairpin ribozyme from cleavage to ligation (Le Vay et al., 2021).

The ability of coacervate phases to strongly partition a wide range of molecular and macromolecular species (Frankel et al., 2016), and to support a variety of complex enzymatic processes (Dora Tang et al., 2015), makes them appealing proto- and artificial cell models in origin-of-life studies (Ghosh et al., 2021a), synthetic biology (van Stevendaal et al., 2021), and modern biology (Yewdall et al., 2021). Simple coacervates that form from oppositely charged polymers have been rigorously investigated for their ability to selectively partition biomolecules and host a variety of different chemistries and reactions in vitro (Yewdall et al., 2021; Nakashima et al., 2019). However, biological condensates formed by protein–protein or protein–nucleic acid interactions are dynamic systems, and their formation, dissolution, and physical properties are subject to spatiotemporal regulation (Ismail et al., 2021). Similarly, in order to realise a truly convincing model proto- or artificial cell, life-like behaviours such as growth, division, and other dynamic, responsive or non-equilibrium processes are essential. Several dynamic and responsive coacervate systems have been established, which are characterised by a phase change in response to environmental stimuli such as light exposure (Martin et al., 2017; Huang et al., 2021; Kubota et al., 2022), changes in temperature (Lu et al., 2021), or pH (Love et al., 2020; Karoui et al., 2021). Furthermore, non-equilibrium environments formed by gas bubbles inside heated rock pores have been shown to drive the growth, fusion, and division of otherwise inert coacervate microdroplets (Ianeselli et al., 2022).

Enzymatic processes that alter the properties of the coacervate components also affect coacervate properties and behaviour. In charge-based condensates, inducing phase change via enzymatic processes has been achieved by, for example, the conversion of ADP into charge-dense ATP (Nakashima et al., 2018), or the alteration of peptide charge state by phosphorylation (Aumiller and Keating, 2016). This has allowed the reversible generation of coacervate droplets by enzymatic networks. In addition, the polymerisation of UDP in U₂₀-spermine coacervates by polynucleotide phosphorylase has been shown to induce transient non-spherical coacervate morphologies (Spoelstra et al., 2020). All these systems depend on the action of proteinaceous enzymes, which presumably emerged later in molecular evolution, perhaps after the first protocells. However, droplets capable of dynamic change via the action of nucleic acid enzymes such as ribozymes have not been previously reported, partially because the catalytic repertoire of these catalysts is limited when compared to their proteinaceous counterparts (Doudna and Lorsch, 2005). Beyond environmental factors such as solution pH and salt concentration (Li et al., 2018), the physical properties and association behaviour of coacervate droplets are determined by factors such as component chain length (Spruijt et al., 2010) and charge density (Neitzel et al., 2021). Of these factors, we noted that polymer chain length could potentially be addressed by the action of a nucleolytic or ligase ribozyme. Thus, in a coacervate composed of RNA and a positively charged polymer, ribozyme-catalysed RNA cleavage or ligation might alter the physical properties or association behaviour of the system if a sufficiently large change in average RNA length were achieved. In particular, we hypothesised that the elongation of the RNA component could lead to the formation of a denser separated phase with altered physical properties (Li et al., 2018; Liu et al., 2020).

In this work, we demonstrate that coacervate microdroplets formed from a ligase ribozyme and lysine-based peptides display reciprocal and synergistic behaviour, in which coacervation enhances and modulates ribozyme activity, and ribozyme activity modulates droplet properties and therefore phenotype. We employ a modified ligase ribozyme that ligates short substrate strands into long concatemers, thus increasing the length of the polyanionic coacervate component. We find that coacervation enhances the rate of substrate ligation 50-fold and inhibits the formation of circular reaction products. In turn, the activity of the ribozyme imparts specific physical properties or phenotypes to the droplets it is contained within, which are not observed in droplets containing inactive ribozymes. These altered behaviours include the inhibition of droplet growth, surface wetting, and content exchange, which could provide fitness advantages under certain conditions. Connecting the sequence information of the ribozyme RNA to the physical properties of the resulting microdroplets is an example of a phenotype–genotype linkage, which is a fundamental requirement for the Darwinian evolution of protocellular systems (Tawfik and Griffiths, 1998).

To investigate the effect of ribozyme activity on coacervate behaviour, we initially sought to design a ribozyme system capable of increasing RNA chain length via concatenation. Although several examples of RNA ligase ribozymes have previously been reported (Paul and Joyce, 2002; Ekland et al., 1995; Hayden et al., 2005), these typically catalyse the ligation of a single junction, which results in only a modest overall increase in average RNA chain length. To achieve greater product lengths, we harnessed the catalytic core of the R3C ligase ribozyme (E_R), whose RNA ligation activity is based on 5′-triphosphate activated substrates (Paul and Joyce, 2002). This system has recently been shown to be active in the presence of poly(L-lysine) under certain conditions, and so was a promising starting point when considering compatibility with coacervate systems (Iglesias-Artola et al., 2022). We previously redesigned the ribozyme–substrate complex to iteratively produce long RNA concatemers from short oligonucleotides (Matreux et al., 2021). In our final design, the ribozyme (E_L) catalyses concatenation of a 31 nt substrate (Figure 1a). Screening of reaction conditions established that strong activity was observed at pH 8.6 in the presence of 10 mM MgCl₂ at a range of temperatures (30, 37, and 45°C) (Figure 1—figure supplement 1). The strongest activity was observed with a 1:1 monomer concentration ratio of substrate:ribozyme, while excess substrate inhibited the formation of longer length products at lower temperatures, most likely because the ribozyme has a higher probability of binding unligated substrates than already growing chains. In order to reduce hydrolytic degradation of RNA, we chose to maximise activity at 30°C with a 1:1 substrate:ribozyme ratio in all subsequent experiments.

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Unedited and uncropped gel image for Figure 1b, and labelled image showing key bands and conditions.

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Numerical turbidity data for Figure 1c.

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Unprocessed and uncropped fluorescence microscope image for 0.75:1 (Lys)_19-72:RNA condensates (Figure 1d) imaged using 10% Cy5-tagged substrate strand.

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Unedited and uncropped gel images for ribozyme kinetics (Figure 1e).

Lane identities are listed in the accompanying spreadsheet.

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The phase separation behaviour of the ribozyme system in the presence of poly(L-lysine) was initially investigated by titrating increasing amounts of (Lys)_19-72 into a fixed concentration of RNA (total monomer concentration = 1 mM) and measuring endpoint activity. The reaction products were separated by urea PAGE and stained using SYBR Gold to visualise all reaction components. The activity of the ribozyme was inhibited in the presence of excess peptide ((Lys)_19-72:RNA > 1) (Figure 1b), but yields of extended products at the endpoint of the reaction (2 hr) were enhanced above the solution reaction at lower ratios (lane profiles, Figure 1b). The reported concentration ratios are calculated from RNA and peptide monomer unit concentrations, and as such are also equivalent to charge ratios. The addition of (Lys)_19-72 led to a gradual increase in turbidity due to phase separation above the critical coacervation concentration of CCC_19-72 ≈ 0.14:1 (Lys)_19-72:RNA (Figure 1c). These experiments were repeated with a shorter peptide ((Lys)_5-24) (Figure 1—figure supplement 2), for which the onset of coacervation occurred at higher peptide:RNA ratios (CCC_5-24 ≈ 0.93:1 (Lys)_5-24:RNA), corroborating previous observations with poly(L-lysine) and the hairpin ribozyme (HPz) (Le Vay et al., 2021). In this case, the activity of the ribozyme was not inhibited in the presence of excess peptide, and again yields of extended products were enhanced above the solution condition (lane profiles, Figure 1—figure supplement 2a). For further experimentation, we selected specific peptide:RNA ratios of 0.75:1 (Lys)_19-72:RNA and 3:1 (Lys)_5-24:RNA, both of which supported coacervation and ribozyme catalysis. Different concentration ratios are required to produce similar droplets for each peptide due to their differing critical coacervation concentrations. Both selected concentration ratios corresponded to points immediately before the plateau in the respective peptide titration turbidity curves and allowed the formation of liquid coacervate droplets without suppression of ribozyme activity. (Figure 1c and Figure 1—figure supplement 2b). Fluorescence imaging of samples at these ratios confirmed the formation of phase-separated coacervate droplets that strongly partitioned the Cy5-labelled RNA substrate (Figure 1d and Figure 1—figure supplement 2c).

To investigate the kinetics of chain elongation and quantify the final product length, we conducted experiments using a 5′-fluorescently labelled substrate to visualise ligation products. At the chosen ratios, ligation rate and product length were greatly enhanced in the coacervate phase compared to solution (Figure 1e and f and Figure 1—figure supplement 3). The kinetic analyses show that the addition of either (Lys)_n peptide resulted in an approximately 50-fold increase in the rate of concatenation and led to the formation of substrate chains with an average length approximately 30 nt greater than those produced in solution (Figure 1e). In solution, the kinetics of chain elongation were best approximated using a first order model (k = 1.5 × 10^–2 ± 1.0 × 10^–3 min^–1), and a final average product length (n) of 52.2 ± 0.5 nt was observed (Figure 1e). In the (Lys)_19-72 coacervate phase, the ribozyme kinetics were best described by a two-phase model (k_fast = 7.7 × 10^–1 ± 0.7 × 10^–1 min^–1, k_slow = 6.6 × 10^–2 ± 1.3 × 10^–2 min^–1), with a final product length of n = 77.8 ± 0.4 nt for (Lys)_19-72. Similar values were obtained for the short peptide (Figure 1—figure supplement 2e, Source data in Figure 1—figure supplement 2—source data 4). We have previously observed a coacervation-induced shift from monophasic to biphasic kinetic behaviour for the hammerhead ribozyme, albeit without an associated increase in rate (Drobot et al., 2018). High local RNA and Mg²⁺ concentrations may lead to enhanced activity by promoting folding and increasing substrate and Mg²⁺ cofactor abundance within the RNA–peptide coacervates (Iglesias-Artola et al., 2022; Frankel et al., 2016). To clarify origin of the enhancement reported here, we performed reactions at varying total RNA concentrations, varying MgCl₂ concentrations and in the presence of crowding agents (Figure 1—figure supplement 4). Briefly, doubling the current total RNA concentration increased product yields, but not to the level observed in the presence of poly(L-lysine), whilst halving the current total RNA concentration reduced product yields (Figure 1—figure supplement 4a). The addition of either PEG or increased MgCl₂ concentrations to the reaction mixture increased reaction yields relative to solution (Figure 1—figure supplement 4b and c), although again not to the same extent as phase separation with poly(L-lysine). We then tested the effect of combinations of PEG and Mg²⁺ on activity (Figure 1—figure supplement 4d) and found that under certain conditions the presence of the crowding agent and additional Mg²⁺ resulted in ligation yields similar to those observed in the RNA-peptide coacervates. These results suggest that multiple factors, including increased concentrations of both RNA and Mg²⁺, are required for enhanced activity in the RNA-peptide coacervates.

In solution, the E_L ribozyme produced both linear and circular concatenates, the latter of which are visible on urea PAGE gels as additional bands visible in between and above the regularly spaced linear ladder of products due to the different mobility of linear and circular products through the gel matrix. The presence of circular products was confirmed by treatment of reacted RNA with RNase R, a 3′ to 5′ exoribonuclease that only digests linear strands (Figure 1—figure supplement 5). The presence of the long peptide (0.75:1 (Lys)_19-72:RNA) suppressed circularisation altogether, whilst the short peptide (3:1 (Lys)_5-24:RNA) reduced the formation of circular products.

Having demonstrated that the E_L ribozyme is capable of substrate concatenation in both solution and in (Lys)_n coacervate droplets, and that its activity is enhanced within the coacervate environment, we aimed to determine whether the elongation of the RNA component would lead to differences in droplet properties, thus altering phenotype. To identify changes due solely to chain concatenation, we developed an inactive mutant of the ribozyme by introducing several point mutations (‘Methods’, Table 1 and Table 2), without altering the substrate binding region. Ribozyme activity assays revealed that the mutant was completely inactive in solution and in the presence of both (Lys)_n peptides at the previously specified charge ratios (Figure 2—figure supplement 1). Populations of droplets containing either the active or inactive ribozyme were loaded into a passivated well plate and imaged over the course of 24 hr. In these experiments, (Lys)_n was added to the RNA reaction mixture immediately, so that all ligation activity would occur in the presence of the peptide. In all cases, phase-separated droplets were observed which persisted over the course of the experiment (Figure 2a and c and Figure 2—figure supplement 2a and c). Whilst many droplets remained static, some coarsening to form larger droplets was observed, particularly in the droplet populations containing the inactive ribozyme. To fully quantify these observations, a segmentation algorithm was used to identify droplets and thereby measure their size and population density (Stringer et al., 2021). Droplets which contained active ribozyme maintained a constant average area over the 24 hr period, whilst droplets containing inactive ribozyme grew, in some cases reaching a final area more than twice that of the active system (Figure 2e and Figure 2—figure supplements 2 and 3). As the average droplet area increased for the inactive droplets, there was a concomitant decrease in droplet population density (the average number of droplets per 100 µm²), indicating that the growth was at least, in part, due to coalescence events between the droplets (Figure 2f). A minimal decrease in droplet population density was observed for active droplets, suggesting that lack of coalescence prevented growth. Similar behaviour was observed for both the long and short peptide (Figure 2—figure supplements 2 and 3), although for the short peptide system, the number of both active and inactive droplets decreased similarly over the course of the experiment, indicating that coalescence was not completely supressed in the active droplets. Intriguingly, some particles formed from the short peptide and active ribozyme system initially adopted a non-spherical morphology and relaxed to form round droplets over the course of the experiment (Figure 2—figure supplement 4), similar to morphological changes induced by the extension of UDP in spermine-based coacervates (Spoelstra et al., 2020). This indicates that the material properties of the droplets change over time.

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Unprocessed and uncropped fluorescence microscope image for 0.75:1 (Lys)_19-72:RNA condensates on passivated surfaces imaged using 10% Cy5-tagged substrate strand.

Extracted numerical parameters are listed in the accompanying spreadsheet.

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Unprocessed and uncropped fluorescence microscope image for 0.75:1 (Lys)_19-72:RNA condensates on unpassivated surfaces imaged using 10% Cy5-tagged substrate strand.

Extracted numerical parameters are listed in the accompanying spreadsheet.

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Whilst surface passivation is used as standard in the imaging of coacervate systems to prevent wetting and adhesion effects, surface interactions can affect droplet formation and morphology, and therefore may also influence the observed protocellular phenotype. Consequently, we repeated the previous experiment on an unpassivated polystyrene surface (Greiner µclear microplate, medium binding). The coacervate droplets containing the active ribozyme behaved as previously observed, with discrete round morphologies (Figure 2b) and little change in both average droplet area and population density over the course of the experiment (Figure 2g and h). These droplets exhibited greater average areas than those in the passivated environment, likely due to wetting onto the surface. In contrast, droplets containing the inactive ribozyme wet the surface and rapidly spread, eventually merging to form a film of the condensed coacervate phase on the bottom of the well (Figure 2d). The measured average particle area therefore increased greatly over the course of the experiment, whilst the number of particles decreased (Figure 2g and h).

These differences in behaviour indicate that the generation of longer RNA within the coacervate environment imparts altered physical properties on the droplets, despite maintaining a spherical morphology typical of a liquid system. To further investigate this phenomenon, we added the (Lys)_5-24 and (Lys)_19-72 peptides to pre-reacted RNA mixtures. Here, we observed that mixtures containing the active ribozyme and therefore pre-concatenated RNA initially formed non-spherical gel-like condensates with both peptides, which relaxed to form spherical droplets over the course of 24 hr, whilst mixtures with inactive ribozyme yielded spherical droplets from the outset (Figure 2—figure supplement 5). This suggests that large morphological differences between active and inactive systems are not observed when peptide is added to ribozyme and substrate mixtures without pre-reaction because the average RNA length is initially identical. As the reaction proceeds in the active systems, a transition to a more viscous or gel-like state may occur whilst maintaining the initially formed spherical morphology.

Given the effect of RNA concatenation on the coacervate droplets, we asked whether the activity of the E_L ribozyme could affect the interactions between different populations of droplets. Using the previously established conditions and concentration ratios, we mixed populations of droplets containing either a Cy5- or FAM-tagged substrate (10% total substrate concentration) and monitored mixing and content exchange over the course of 24 hr. These effects can be quantified by the calculation of a Pearson correlation coefficient (PCC), which measures the correlation of pixel intensities between the two fluorescence channels (Dunn et al., 2011). Two coefficients were calculated: PCC_droplet, which measures the degree to which fluorophores mix within individual fused droplets, and PCC_pop, which is calculated on a population level and measures the degree of mixing between the two populations. Positive values indicate spatial colocalisation of fluorophores, whilst negative values indicate spatial separation of fluorophores and therefore the presence of discrete regions or populations.

Shortly following mixing, discrete populations of Cy5- and FAM-labelled droplets are clearly visible (Figure 3a and b and Figure 3—figure supplement 1). Fused and unevenly mixed droplets containing both fluorophores are visible, providing evidence that coalescence is the mechanism of droplet growth in this system. After 24 hr, visual inspection of the images reveals that discrete populations of Cy5- and FAM-labelled droplets only persist in droplets containing the active ribozyme (Figure 3a and Figure 3—figure supplement 1a). In the inactive droplets, both fluorophores appear evenly distributed throughout the population (Figure 3b and Figure 3—figure supplement 1b). In all cases, the PCC_droplet increased over time, tending towards unity, indicating that labelled RNA was able to equilibrate within the droplets via diffusion (Figure 3c and Figure 3—figure supplement 1c). However, it is notable that the rate and magnitude of increase in PCC_droplet was greater in droplets containing the inactive ribozyme (Figure 3—figure supplement 2), indicating greater RNA mobility. For active and inactive (Lys)_19-72 droplets, PCC_pop was initially negative, implying discrete and distinct populations (Figure 3d). Over the course of the experiment, the active system maintained this separation, whilst the PCC_pop of the inactive system tended towards unity. These trends are further confirmed by scatter plots of normalised FAM and Cy5 fluorescence intensity in individual droplets (Figure 3—figure supplement 3), which show the clearly separated populations of active systems and mixing in inactive systems. As observed in our previous experiments, mixtures of the active ribozyme and peptide produced smaller droplets which did not grow over the course of the experiment compared to inactive populations, suggesting that droplets with inactive ribozyme are more prone to coalesce and therefore mixing than droplets containing the active ribozyme (Figure 3e and f).

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Unprocessed and uncropped fluorescence microscope images of mixed populations of 0.75:1 (Lys)_19-72:RNA condensates containing active ribozyme imaged in both FAM and Cy5 channels.

Extracted numerical parameters are listed in the accompanying spreadsheet.

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Unprocessed and uncropped fluorescence microscope images of mixed populations of 0.75:1 (Lys)_19-72:RNA condensates containing inactive ribozyme imaged in both FAM and Cy5 channels.

Extracted numerical parameters are listed in the accompanying spreadsheet.

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A similar yet weaker trend was observed with short (Lys)_5-24 peptide droplets, which exhibited greater content exchange overall between both active and inactive droplets (Figure 3—figure supplements 1–3). Here, the presence of the active ribozyme slowed the rate of coalescence and therefore content exchange rather than completely suppressing it, but still allowed active populations to maintain a limited degree of identity over the course of the experiment.

Our original goal was to develop a ribozyme capable of RNA chaining that could significantly increase the average RNA length in a coacervate system, and thereby allow us to study the influence of such a reaction on the material properties of the coacervates. Although ribozyme systems that concatenate a short substrate have been previously reported, these do so via a reversible cyclic-phosphate-mediated mechanism, and as such the concentration of extended products decreases rapidly as product length increases (Mutschler et al., 2015). The R3C ligase ribozyme was thus an attractive starting point for the development of our system as the 5′-triphosphate activated reaction it catalyses is quasi-irreversible, and the ribozyme is already optimised for high rate and yield (Paul and Joyce, 2002). Indeed, the E_L ribozyme was able to generate RNA strands of >500 nt in length, including circular species, and this activity was enhanced in terms of both rate and yield in (Lys)_n coacervates. Although the observation of circular products in concatenation reactions has been previously reported in ribozyme systems (Jeancolas et al., 2021), the inhibition of this behaviour by coacervation is notable. Whilst the mechanism of this inhibition is unknown, the absence of circular products suggests that the ends of a substrate strand do not meet, hinting at a decrease in RNA mobility, increased chain stiffness, or reduced release of ligated substrate from the ribozyme within the condensed phase compared to the bulk solution. The suppression of circularisation should lead to higher overall RNA lengths as the concentration of RNA ends available for ligation is not decreased over the course of the reaction. It is challenging to quantify this difference under the current experimental setup, as measurement of the average chain lengths requires the use of a 5′ fluorescent tag (as in the kinetics experiment), which itself prevents formation of circular products due to the blocked 5′ end. A potential solution to overcome this limitation would be to use a substrate with an internal fluorophore (assuming it does not affect ligation activity) or a radiolabel.

We have previously reported that the activity of the hairpin ribozyme (HPz) is greatly enhanced in condensed phases comprised of catalytic RNA and (Lys)_n peptides (Le Vay et al., 2021). The increase in turbidity observed on titrating either (Lys)_19-72 or (Lys)_5-24 into a fixed concentration of R3C ribozyme and substrate was similar to that observed for HPz, although in this study the resultant condensed phase appeared liquid rather than gel-like. This variation in condensed phase morphology may be due to the influence of RNA structure, sequence, and hybridisation state on condensation (Vieregg et al., 2018; Boeynaems et al., 2019), with a higher degree of single-stranded or loop structures in the E_L ribozyme system. The inhibition of ribozyme activity at excess (Lys)_19-72:RNA ratios was comparable to that observed in our previous study (Le Vay et al., 2021) and is attributed to a peptide length-dependent melting or misfolding of native nucleic acid tertiary structures at high concentrations of longer peptides (Cakmak et al., 2020; Nott et al., 2016). A previous study into the function of the R3C replicase ribozyme in RNA–peptide coacervates reported a similar trend: catalytic activity was inhibited at excess (Lys)_n:RNA ratios for a range of peptides (n = 7, 8, 9, 10, 18, and 19–72) (Iglesias-Artola et al., 2022). The present study extends the peptide concentration range in which the R3C ribozyme functions, with robust catalytic activity at ratios as high as 4:1 (Lys)_5-24:RNA. A possible explanation is that commercially available (Lys)_n deviates from the manufacturer’s stated size range, with the (Lys)_5-24 used here being predominantly comprised of oligomers between n = 3 to n = 9 in length (Le Vay et al., 2021). These short fragments may interact only weakly with the RNA, effectively reducing the concentration of peptide oligomers that can form condensates. In general, it should be noted that the effect of phase separation on ribozyme activity in a given system is highly specific to the identity of the ribozyme and peptide, their relative concentrations, and environmental conditions such as buffer, magnesium concentration, and temperature.

The difference in growth behaviour between active and inactive droplets is a striking demonstration of how the presence of a catalytic species can modulate the physical properties and behaviour of a model protocellular system, in this case by reducing coalescence. The coarsening of liquid coacervate phases is not inevitable: growth can be also be suppressed by active chemical processes hosted within droplets that produce the polymer components (Zwicker et al., 2015), although in our experiments the action of the ribozyme does not increase the concentration of RNA or peptide in the system. The formation of kinetically trapped states can also allow the persistence of static droplet populations over time (Ranganathan and Shakhnovich, 2020). However, increasing polymer lengths in a coacervate system results in stronger cooperative electrostatic interactions between polymer components (Li et al., 2018), and has been shown to affect physical parameters such as droplet water content, critical salt concentration, and condensed phase polymer concentration (Spruijt et al., 2010), leading to denser droplets and a more depleted dilute phase. Simulations have shown that increasing RNA length modulates both the material and interfacial properties of RNA–peptide condensates, in particular increasing density and surface tension (Laghmach et al., 2022). RNA length also has been shown to modulate viscosity in biomolecular condensates (Tejedor et al., 2021), which in turn determines the droplet fusion dynamics (Ghosh et al., 2021b). Thus, increasing overall RNA length by R3C substrate concatenation may inhibit growth via coalescence by increasing condensate density and viscosity, and reduce wetting affects through the alteration of surface tension. Whilst RNA sequence and secondary structure have also been shown to influence the viscoelastic behaviour of RNA peptide condensates (Alshareedah et al., 2021), the active and inactive ribozyme systems used in the present study differ by only few nucleotides.

Despite the spherical morphology of the RNA–peptide condensates reported here, the inhibition of growth and wetting may also be due to a phase transition to a gel state. The formation of non-liquid RNA–peptide condensates has been previously reported for poly-rA:rU-peptide mixtures, where RNA base pairing leads to the formation of a kinetically arrested gel-like solid (Boeynaems et al., 2019), as well as in our previous work (Le Vay et al., 2021). These gel condensates melt to form spherical particles upon thermal denaturation and annealing, which disrupts the networked structures formed from complementary RNA strands and peptides. In our R3C system, complementary base-pairing interactions between substrate and ribozyme should also permit the formation of large, non-covalently assembled networked structures due to the repeating nature of the substrate. Ribozyme activity is expected to increase the stability of these structures as substrate ligation increases the free energy of association between ribozyme and substrate. We hypothesise that in the inactive system, and before significant concatenation occurs in the active system, RNA–RNA interactions are sufficiently weak to allow the formation of liquid coacervate droplets. In the active system, liquid droplets initially form because the timescale of coalescence is faster than that of RNA ligation. However, as ligation proceeds the stability of networked assemblies of ribozyme and substrate increases, resulting in a transition from a liquid to either a highly viscous liquid state or solid gel state that is both unable to grow and or wet the unpassivated surface on the timescale of the experiment. This hypothesis is supported by our observation that mixtures of pre-concatenated RNA and (Lys)_n initially form non-spherical gel-like aggregates, which then relax to form spherical droplets over the course of 24 hr.

The reduction of mixing between populations of droplets containing orthogonally labelled RNA likely results from the changes in material properties and growth behaviour caused by RNA concatenation. The coalescence of the coacervate droplets observed in all inactive systems is likely to be the primary mechanism of mixing in this scenario. Indeed, greater droplet growth was observed in the two-population system than for single-population experiments, which may be due to the necessity of pipetting each droplet population into the sample environment sequentially, thus increasing mechanical agitation and contacts between droplets. All populations at the initial time point contain fusion droplets, which contain separate and unmixed areas of each fluorescent RNA. Notably, fusion droplets in all systems appear evenly mixed after 24 hr, suggesting that significant equilibration occurs before any transition to a highly viscous liquid or solid gel. Mixing may also occur by diffusive transfer of RNA between droplets. As the fluorescently labelled substrate is ligated onto the growing concatemer chain, the diffusion coefficient of the labelled material is expected to decrease over the course of the reaction as its effective length increases, which would in turn slow diffusive transfer in the active system.

Taken together, the results reported here describe a range of altered droplet behaviours stemming from ribozyme-driven changes in coacervate physical properties. These physical changes may impact other processes relevant to early compartmentalised replicators and protocells, such as resistance to molecular parasites (Koonin et al., 2017) and metabolite exchange (Smokers et al., 2022), and thus are exciting avenues for further study. The ability of a population of protocells to resist passive coarsening, surface wetting, and content exchange can already be considered a fitness advantage (Nakashima et al., 2021), and the emergence of fitness differences from varying protocellular compositions and phenotypes is a key step on the path towards Darwinian evolution with suitable selective pressures (Adamala and Szostak, 2013). Furthermore, in this system the phenotypic difference originates from the ribozyme-driven concatenation reaction, which increases the length of the anionic coacervate component. The sequence of the ribozyme in effect comprises the genotype of our protocellular system, with different variants (e.g. active or inactive sequences) producing different protocellular phenotypes. The realisation of a phenotype–genotype linkage is essential for true open-ended evolution, otherwise phenotypes with obvious fitness advantages have no possibility of being inherited by future generations. We envision that recent methodological developments in RNA sequencing from single coacervate microdroplets will enable future selection experiments on populations of protocells with varying RNA genotypes and varying degrees of fitness with respect to environmental pressures such as heat or salt concentration (Wollny et al., 2022).

The reciprocal modulation of ribozyme activity and coacervate properties reported here furthers the argument for the early coevolution of RNA and peptides (Frenkel-Pinter et al., 2020; Tagami et al., 2017; Seal et al., 2022; Ghosh et al., 2021a; Le Vay and Mutschler, 2019). However, further development is required to move towards more realistic model systems. A key limitation is that the droplets presented in this work contain a large proportion or pure ribozyme and substrate, which would presumably be less abundant in a more plausible scenario. The R3C ligase is a highly optimised system developed by in vitro selection and offers little potential for further enhancement of catalytic activity, but similar material changes with less ribozyme might be achieved by optimising the substrate binding arms and conditions for multiturnover reaction, or tuning substrate length to produce greater overall changes with fewer ligations. We have previously demonstrated substrate strand release in hairpin ribozyme-(Lys)_n condensates (Le Vay et al., 2021), so it may be possible to engineer similar behaviour in the R3C system, perhaps at the cost of reaction rate. In addition, the simple lysine polypeptides used here are a very basic model system, but additional variation in droplet properties may be achieved in future by varying peptide sequence. Droplet populations formed from different peptides could exhibit varying degrees of ribozyme enhancement or fundamentally different physical properties that allow selection based on peptide identity as well as RNA sequence and activity. Although the magnitude of behavioural change was greater for coacervate populations formed with the longer peptide, clear phenotypic differences were nonetheless also present in the (Lys)_5-24 system. This short peptide, predominantly composed of n = 3–9 residue oligomers, is of a length that could be produced by prebiotically plausible processes such as wet–dry cycling (Forsythe et al., 2015; Rodriguez-Garcia et al., 2015).

All data generated during this study are included in the manuscript and supporting files; source data files have been provided for all figures and figure supplements.

1. 1. Abbas M
   2. Lipiński WP
   3. Wang J
   4. Spruijt E

   (2021) Peptide-based coacervates as biomimetic protocells

   Chemical Society Reviews 50:3690–3705.

   https://doi.org/10.1039/d0cs00307g

   • PubMed
   • Google Scholar
2. 1. Adamala K
   2. Szostak JW

   (2013) Competition between model Protocells driven by an encapsulated catalyst

   Nature Chemistry 5:495–501.

   https://doi.org/10.1038/nchem.1650

   • PubMed
   • Google Scholar
3. 1. Alshareedah I
   2. Moosa MM
   3. Pham M
   4. Potoyan DA
   5. Banerjee PR

   (2021) Programmable viscoelasticity in protein-RNA condensates with disordered sticker-spacer Polypeptides

   Nature Communications 12:6620.

   https://doi.org/10.1038/s41467-021-26733-7

   • PubMed
   • Google Scholar
4. 1. Aumiller WM
   2. Keating CD

   (2016) Phosphorylation-mediated RNA/peptide complex Coacervation as a model for intracellular liquid organelles

   Nature Chemistry 8:129–137.

   https://doi.org/10.1038/nchem.2414

   • PubMed
   • Google Scholar
5. 1. Boeynaems S
   2. Holehouse AS
   3. Weinhardt V
   4. Kovacs D
   5. Van Lindt J
   6. Larabell C
   7. Van Den Bosch L
   8. Das R
   9. Tompa PS
   10. Pappu RV
   11. Gitler AD

   (2019) Spontaneous driving forces give rise to protein-RNA condensates with Coexisting phases and complex material properties

   PNAS 116:7889–7898.

   https://doi.org/10.1073/pnas.1821038116

   • PubMed
   • Google Scholar
6. 1. Cakmak FP
   2. Choi S
   3. Meyer MO
   4. Bevilacqua PC
   5. Keating CD

   (2020) Prebiotically-relevant low Polyion Multivalency can improve Functionality of Membraneless compartments

   Nature Communications 11:5949.

   https://doi.org/10.1038/s41467-020-19775-w

   • PubMed
   • Google Scholar
7. 1. Dora Tang TY
   2. van Swaay D
   3. deMello A
   4. Ross Anderson JL
   5. Mann S

   (2015) In vitro gene expression within membrane-free Coacervate Protocells

   Chemical Communications 51:11429–11432.

   https://doi.org/10.1039/c5cc04220h

   • PubMed
   • Google Scholar
8. 1. Doudna JA
   2. Lorsch JR

   (2005) Ribozyme Catalysis: not different, just worse

   Nature Structural & Molecular Biology 12:395–402.

   https://doi.org/10.1038/nsmb932

   • Google Scholar
9. 1. Drobot B
   2. Iglesias-Artola JM
   3. Le Vay K
   4. Mayr V
   5. Kar M
   6. Kreysing M
   7. Mutschler H
   8. Tang T-YD

   (2018) Compartmentalised RNA catalysis in membrane-free coacervate protocells

   Nature Communications 9:3643.

   https://doi.org/10.1038/s41467-018-06072-w

   • PubMed
   • Google Scholar
10. 1. Dunn KW
    2. Kamocka MM
    3. McDonald JH

    (2011) A practical guide to evaluating Colocalization in biological microscopy

    American Journal of Physiology. Cell Physiology 300:C723–C742.

    https://doi.org/10.1152/ajpcell.00462.2010

    • PubMed
    • Google Scholar
11. 1. Ekland EH
    2. Szostak JW
    3. Bartel DP

    (1995) Structurally complex and highly active RNA Ligases derived from random RNA sequences

    Science 269:364–370.

    https://doi.org/10.1126/science.7618102

    • PubMed
    • Google Scholar
12. 1. Forsythe JG
    2. Yu S-S
    3. Mamajanov I
    4. Grover MA
    5. Krishnamurthy R
    6. Fernández FM
    7. Hud NV

    (2015) Ester‐Mediated amide bond formation driven by wet–dry cycles: A possible path to Polypeptides on the Prebiotic earth

    Angewandte Chemie 54:9871–9875.

    https://doi.org/10.1002/anie.201503792

    • PubMed
    • Google Scholar
13. 1. Frankel EA
    2. Bevilacqua PC
    3. Keating CD

    (2016) Polyamine/nucleotide coacervates provide strong compartmentalization of Mg2+, nucleotides, and RNA

    Langmuir 32:2041–2049.

    https://doi.org/10.1021/acs.langmuir.5b04462

    • PubMed
    • Google Scholar
14. 1. Frenkel-Pinter M
    2. Haynes JW
    3. Mohyeldin AM
    4. Martin C
    5. Sargon AB
    6. Petrov AS
    7. Krishnamurthy R
    8. Hud NV
    9. Williams LD
    10. Leman LJ

    (2020) Mutually stabilizing interactions between proto-peptides and RNA

    Nature Communications 11:3137.

    https://doi.org/10.1038/s41467-020-16891-5

    • PubMed
    • Google Scholar
15. 1. Ghosh B
    2. Bose R
    3. Tang TYD

    (2021a) Can Coacervation unify disparate hypotheses in the origin of cellular life

    Current Opinion in Colloid & Interface Science 52:101415.

    https://doi.org/10.1016/j.cocis.2020.101415

    • Google Scholar
16. 1. Ghosh A
    2. Kota D
    3. Zhou HX

    (2021b) Shear relaxation governs fusion Dynamics of Biomolecular condensates

    Nature Communications 12:5995.

    https://doi.org/10.1038/s41467-021-26274-z

    • PubMed
    • Google Scholar
17. 1. Hayden EJ
    2. Riley CA
    3. Burton AS
    4. Lehman N

    (2005) RNA-directed construction of structurally complex and active Ligase Ribozymes through Recombination

    RNA 11:1678–1687.

    https://doi.org/10.1261/rna.2125305

    • PubMed
    • Google Scholar
18. 1. Huang Y
    2. Wang X
    3. Li J
    4. Lin Y
    5. Chen H
    6. Liu X
    7. Huang X

    (2021) Reversible light-responsive Coacervate Microdroplets with rapid regulation of enzymatic reaction rate

    ChemSystemsChem 3:e2100006.

    https://doi.org/10.1002/syst.202100006

    • Google Scholar
19. 1. Hyman AA
    2. Weber CA
    3. Jülicher F

    (2014) Liquid-liquid phase separation in biology

    Annual Review of Cell and Developmental Biology 30:39–58.

    https://doi.org/10.1146/annurev-cellbio-100913-013325

    • PubMed
    • Google Scholar
20. 1. Ianeselli A
    2. Tetiker D
    3. Stein J
    4. Kühnlein A
    5. Mast CB
    6. Braun D
    7. Dora Tang T-Y

    (2022) Non-equilibrium conditions inside rock pores drive fission, maintenance and selection of Coacervate Protocells

    Nature Chemistry 14:32–39.

    https://doi.org/10.1038/s41557-021-00830-y

    • PubMed
    • Google Scholar
21. 1. Iglesias-Artola JM
    2. Drobot B
    3. Kar M
    4. Fritsch AW
    5. Mutschler H
    6. Dora Tang T-Y
    7. Kreysing M

    (2022) Charge-density reduction promotes Ribozyme activity in RNA–peptide Coacervates via RNA Fluidization and magnesium partitioning

    Nature Chemistry 14:407–416.

    https://doi.org/10.1038/s41557-022-00890-8

    • PubMed
    • Google Scholar
22. 1. Ismail H
    2. Liu X
    3. Yang F
    4. Li J
    5. Zahid A
    6. Dou Z
    7. Liu X
    8. Yao X

    (2021) Mechanisms and regulation underlying Membraneless Organelle plasticity control

    Journal of Molecular Cell Biology 13:239–258.

    https://doi.org/10.1093/jmcb/mjab028

    • PubMed
    • Google Scholar
23. 1. Jeancolas C
    2. Matsubara YJ
    3. Vybornyi M
    4. Lambert CN
    5. Blokhuis A
    6. Alline T
    7. Griffiths AD
    8. Ameta S
    9. Krishna S
    10. Nghe P

    (2021) RNA diversification by a self-reproducing Ribozyme revealed by deep sequencing and kinetic Modelling

    Chemical Communications 57:7517–7520.

    https://doi.org/10.1039/D1CC02290C

    • Google Scholar
24. 1. Karoui H
    2. Seck MJ
    3. Martin N

    (2021) Self-programmed enzyme phase separation and Multiphase Coacervate Droplet organization

    Chemical Science 12:2794–2802.

    https://doi.org/10.1039/d0sc06418a

    • PubMed
    • Google Scholar
25. 1. Koonin EV
    2. Wolf YI
    3. Katsnelson MI

    (2017) Inevitability of the emergence and persistence of genetic parasites caused by evolutionary instability of parasite-free States

    Biology Direct 12:31.

    https://doi.org/10.1186/s13062-017-0202-5

    • PubMed
    • Google Scholar
26. 1. Kubota R
    2. Torigoe S
    3. Hamachi I

    (2022) Temporal stimulus patterns drive differentiation of a synthetic Dipeptide-based Coacervate

    Journal of the American Chemical Society 144:15155–15164.

    https://doi.org/10.1021/jacs.2c05101

    • PubMed
    • Google Scholar
27. 1. Laghmach R
    2. Alshareedah I
    3. Pham M
    4. Raju M
    5. Banerjee PR
    6. Potoyan DA

    (2022) RNA chain length and Stoichiometry govern surface tension and stability of protein-RNA condensates

    IScience 25:104105.

    https://doi.org/10.1016/j.isci.2022.104105

    • PubMed
    • Google Scholar
28. 1. Le Vay K
    2. Mutschler H

    (2019) The difficult case of an RNA-only origin of life

    Emerging Topics in Life Sciences 3:469–475.

    https://doi.org/10.1042/ETLS20190024

    • PubMed
    • Google Scholar
29. 1. Le Vay K
    2. Song EY
    3. Ghosh B
    4. Tang T-YD
    5. Mutschler H

    (2021) Enhanced ribozyme-catalyzed recombination and oligonucleotide assembly in peptide-RNA condensates

    Angewandte Chemie 60:26096–26104.

    https://doi.org/10.1002/anie.202109267

    • PubMed
    • Google Scholar
30. 1. Li L
    2. Srivastava S
    3. Andreev M
    4. Marciel AB
    5. de Pablo JJ
    6. Tirrell MV

    (2018) Phase behavior and salt partitioning in Polyelectrolyte complex Coacervates

    Macromolecules 51:2988–2995.

    https://doi.org/10.1021/acs.macromol.8b00238

    • Google Scholar
31. 1. Liu Y
    2. Santa Chalarca CF
    3. Carmean RN
    4. Olson RA
    5. Madinya J
    6. Sumerlin BS
    7. Sing CE
    8. Emrick T
    9. Perry SL

    (2020) Effect of polymer chemistry on the linear viscoelasticity of complex Coacervates

    Macromolecules 53:7851–7864.

    https://doi.org/10.1021/acs.macromol.0c00758

    • Google Scholar
32. 1. Love C
    2. Steinkühler J
    3. Gonzales DT
    4. Yandrapalli N
    5. Robinson T
    6. Dimova R
    7. Tang TYD

    (2020) Reversible pH responsive Coacervate formation in lipid Vesicles activates dormant enzymatic reactions

    Angewandte Chemie 59:5950–5957.

    https://doi.org/10.1002/anie.201914893

    • PubMed
    • Google Scholar
33. 1. Lu T
    2. Nakashima KK
    3. Spruijt E

    (2021) Temperature-responsive peptide-nucleotide Coacervates

    The Journal of Physical Chemistry B 125:3080–3091.

    https://doi.org/10.1021/acs.jpcb.0c10839

    • Google Scholar
34. 1. Martin N
    2. Sharma KP
    3. Harniman RL
    4. Richardson RM
    5. Hutchings RJ
    6. Alibhai D
    7. Li M
    8. Mann S

    (2017) Light-induced dynamic shaping and self-division of Multipodal Polyelectrolyte-surfactant Microarchitectures via Azobenzene Photomechanics

    Scientific Reports 7:1–12.

    https://doi.org/10.1038/srep41327

    • Google Scholar
35. 1. Matreux T
    2. Le Vay K
    3. Schmid A
    4. Aikkila P
    5. Belohlavek L
    6. Çalışkanoğlu AZ
    7. Salibi E
    8. Kühnlein A
    9. Springsklee C
    10. Scheu B
    11. Dingwell DB
    12. Braun D
    13. Mutschler H
    14. Mast CB

    (2021) Heat flows in rock cracks naturally optimize salt compositions for Ribozymes

    Nature Chemistry 13:1038–1045.

    https://doi.org/10.1038/s41557-021-00808-w

    • PubMed
    • Google Scholar
36. 1. Mutschler H
    2. Wochner A
    3. Holliger P

    (2015) Freeze-thaw cycles as drivers of complex Ribozyme assembly

    Nature Chemistry 7:502–508.

    https://doi.org/10.1038/nchem.2251

    • PubMed
    • Google Scholar
37. 1. Nakashima KK
    2. Baaij JF
    3. Spruijt E

    (2018) Reversible generation of Coacervate droplets in an enzymatic network

    Soft Matter 14:361–367.

    https://doi.org/10.1039/c7sm01897e

    • PubMed
    • Google Scholar
38. 1. Nakashima KK
    2. Vibhute MA
    3. Spruijt E

    (2019) Biomolecular chemistry in liquid phase separated compartments

    Frontiers in Molecular Biosciences 6:21.

    https://doi.org/10.3389/fmolb.2019.00021

    • PubMed
    • Google Scholar
39. 1. Nakashima KK
    2. van Haren MHI
    3. André AAM
    4. Robu I
    5. Spruijt E

    (2021) Active Coacervate droplets are Protocells that grow and resist Ostwald ripening

    Nature Communications 12:3819.

    https://doi.org/10.1038/s41467-021-24111-x

    • PubMed
    • Google Scholar
40. 1. Neitzel AE
    2. Fang YN
    3. Yu B
    4. Rumyantsev AM
    5. de Pablo JJ
    6. Tirrell MV

    (2021) Polyelectrolyte complex Coacervation across a broad range of charge densities

    Macromolecules 54:6878–6890.

    https://doi.org/10.1021/acs.macromol.1c00703

    • PubMed
    • Google Scholar
41. 1. Nott TJ
    2. Craggs TD
    3. Baldwin AJ

    (2016) Membraneless organelles can melt nucleic acid Duplexes and act as Biomolecular filters

    Nature Chemistry 8:569–575.

    https://doi.org/10.1038/nchem.2519

    • PubMed
    • Google Scholar
42. Book

    1. Oparin AI

    (1965) The Origin of Life and the Origin of Enzymes

    John Wiley & Sons, Ltd.

    https://doi.org/10.1002/9780470122723

    • Google Scholar
43. 1. Paul N
    2. Joyce GF

    (2002) A self-replicating Ligase Ribozyme

    PNAS 99:12733–12740.

    https://doi.org/10.1073/pnas.202471099

    • PubMed
    • Google Scholar
44. 1. Poudyal RR
    2. Guth-Metzler RM
    3. Veenis AJ
    4. Frankel EA
    5. Keating CD
    6. Bevilacqua PC

    (2019a) Template-directed RNA polymerization and enhanced Ribozyme Catalysis inside Membraneless compartments formed by Coacervates

    Nature Communications 10:490.

    https://doi.org/10.1038/s41467-019-08353-4

    • PubMed
    • Google Scholar
45. 1. Poudyal RR
    2. Keating CD
    3. Bevilacqua PC

    (2019b) Polyanion-assisted ribozyme catalysis inside complex coacervates

    ACS Chemical Biology 14:1243–1248.

    https://doi.org/10.1021/acschembio.9b00205

    • PubMed
    • Google Scholar
46. 1. Ranganathan S
    2. Shakhnovich EI

    (2020) Dynamic Metastable long-living droplets formed by sticker-spacer proteins

    eLife 9:e56159.

    https://doi.org/10.7554/eLife.56159

    • PubMed
    • Google Scholar
47. 1. Roden C
    2. Gladfelter AS

    (2021) RNA contributions to the form and function of Biomolecular condensates

    Nature Reviews. Molecular Cell Biology 22:183–195.

    https://doi.org/10.1038/s41580-020-0264-6

    • PubMed
    • Google Scholar
48. 1. Rodriguez-Garcia M
    2. Surman AJ
    3. Cooper GJT
    4. Suárez-Marina I
    5. Hosni Z
    6. Lee MP
    7. Cronin L

    (2015) Formation of Oligopeptides in high yield under simple programmable conditions

    Nature Communications 6:8385.

    https://doi.org/10.1038/ncomms9385

    • PubMed
    • Google Scholar
49. 1. Seal M
    2. Weil-Ktorza O
    3. Despotović D
    4. Tawfik DS
    5. Levy Y
    6. Metanis N
    7. Longo LM
    8. Goldfarb D

    (2022) Peptide-RNA Coacervates as a cradle for the evolution of folded domains

    Journal of the American Chemical Society 144:14150–14160.

    https://doi.org/10.1021/jacs.2c11057

    • PubMed
    • Google Scholar
50. Software

    1. Sharma VP

    (2018) Imagej Plugin Hyperstackreg V5.6

    Zenodo.

    https://doi.org/10.5281/zenodo.2252521
51. 1. Smokers IBA
    2. van Haren MHI
    3. Lu T
    4. Spruijt E

    (2022) Complex Coacervation and Compartmentalized conversion of Prebiotically relevant metabolites

    ChemSystemsChem 4:e202200004.

    https://doi.org/10.1002/syst.202200004

    • Google Scholar
52. 1. Spoelstra WK
    2. van der Sluis EO
    3. Dogterom M
    4. Reese L

    (2020) Nonspherical Coacervate shapes in an enzyme-driven active system

    Langmuir 36:1956–1964.

    https://doi.org/10.1021/acs.langmuir.9b02719

    • PubMed
    • Google Scholar
53. 1. Spruijt E
    2. Westphal AH
    3. Borst JW
    4. Cohen Stuart MA
    5. van der Gucht J

    (2010) Binodal compositions of Polyelectrolyte complexes

    Macromolecules 43:6476–6484.

    https://doi.org/10.1021/ma101031t

    • Google Scholar
54. 1. Stringer C
    2. Wang T
    3. Michaelos M
    4. Pachitariu M

    (2021) Cellpose: a generalist algorithm for cellular Segmentation

    Nature Methods 18:100–106.

    https://doi.org/10.1038/s41592-020-01018-x

    • PubMed
    • Google Scholar
55. 1. Tagami S
    2. Attwater J
    3. Holliger P

    (2017) Simple peptides derived from the Ribosomal core potentiate RNA polymerase Ribozyme function

    Nature Chemistry 9:325–332.

    https://doi.org/10.1038/nchem.2739

    • PubMed
    • Google Scholar
56. 1. Tawfik DS
    2. Griffiths AD

    (1998) Man-made cell-like compartments for molecular evolution

    Nature Biotechnology 16:652–656.

    https://doi.org/10.1038/nbt0798-652

    • PubMed
    • Google Scholar
57. 1. Tejedor AR
    2. Garaizar A
    3. Ramírez J
    4. Espinosa JR

    (2021) Rnaa modulation of transport properties and stability in phase-separated condensates

    Biophysical Journal 120:5169–5186.

    https://doi.org/10.1016/j.bpj.2021.11.003

    • PubMed
    • Google Scholar
58. 1. Ukmar-Godec T
    2. Hutten S
    3. Grieshop MP
    4. Rezaei-Ghaleh N
    5. Cima-Omori MS
    6. Biernat J
    7. Mandelkow E
    8. Söding J
    9. Dormann D
    10. Zweckstetter M

    (2019) Lysine/RNA-interactions drive and regulate Biomolecular condensation

    Nature Communications 10:2909.

    https://doi.org/10.1038/s41467-019-10792-y

    • PubMed
    • Google Scholar
59. 1. van Stevendaal MHME
    2. Vasiukas L
    3. Yewdall NA
    4. Mason AF
    5. van Hest JCM

    (2021) Engineering of Biocompatible Coacervate-based synthetic cells

    ACS Applied Materials & Interfaces 13:7879–7889.

    https://doi.org/10.1021/acsami.0c19052

    • PubMed
    • Google Scholar
60. 1. Vieregg JR
    2. Lueckheide M
    3. Marciel AB
    4. Leon L
    5. Bologna AJ
    6. Rivera JR
    7. Tirrell MV

    (2018) Oligonucleotide-peptide complexes: phase control by hybridization

    Journal of the American Chemical Society 140:1632–1638.

    https://doi.org/10.1021/jacs.7b03567

    • PubMed
    • Google Scholar
61. 1. Wang B
    2. Zhang L
    3. Dai T
    4. Qin Z
    5. Lu H
    6. Zhang L
    7. Zhou F

    (2021) Liquid-liquid phase separation in human health and diseases

    Signal Transduction and Targeted Therapy 6:290.

    https://doi.org/10.1038/s41392-021-00678-1

    • PubMed
    • Google Scholar
62. 1. Wollny D
    2. Vernot B
    3. Wang J
    4. Hondele M
    5. Safrastyan A
    6. Aron F
    7. Micheel J
    8. He Z
    9. Hyman A
    10. Weis K
    11. Camp JG
    12. Tang TYD
    13. Treutlein B

    (2022) Characterization of RNA content in individual phase-separated Coacervate Microdroplets

    Nature Communications 13:2626.

    https://doi.org/10.1038/s41467-022-30158-1

    • PubMed
    • Google Scholar
63. 1. Yewdall NA
    2. André AAM
    3. Lu T
    4. Spruijt E

    (2021) Coacervates as models of Membraneless organelles

    Current Opinion in Colloid & Interface Science 52:101416.

    https://doi.org/10.1016/j.cocis.2020.101416

    • Google Scholar
64. 1. Zwicker D
    2. Hyman AA
    3. Jülicher F

    (2015) Suppression of Ostwald ripening in active emulsions

    Physical Review. E, Statistical, Nonlinear, and Soft Matter Physics 92:012317.

    https://doi.org/10.1103/PhysRevE.92.012317

    • PubMed
    • Google Scholar

Decision letter after peer review:

Thank you for submitting your article "Ribozyme-phenotype coupling in peptide-based coacervate protocells" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by Timothy Nilsen as the Reviewing Editor and James Manley as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission. (Please note that point 3 should have read: Paragraphs 3-5 of reviewer 2's author recommendations.)

Essential revisions:

The reviewers were generally quite positive about the work. They have made a number of suggestions that may be useful for future investigation in this area. Of these, some of these are essential as detailed below. We are not asking for any additional experiments, but please discuss the points raised by the reviewers.

1. Clarification of the title.

2. The results shown in figure 1b, 1d are discordant and this needs to be remedied by either choosing different images or (not as good) explaining in the text why these panels seem to be discordant.

3. Paragraphs 3-5 of reviewer 2's author recommendations.

Reviewer #1 (Recommendations for the authors):

1. As the products of the ligation reaction by the EL R3C ladder ribozyme are mixtures of RNAs with different lengths, the exact requirements (e.g., average/maximum length of the ligated RNAs + double strand formation) for the phenotypic changes are not clear. Is S2 or S3 enough? Are longer products (e.g., S6-S17) more important? Do the elongated RNAs need to remain hybridized with the ribozymes? Droplet formation experiments with S2 or S3 (synthesized chemically or by T7 RNAP) in the presence and absence of the inactive hybridizing ribozyme would clarify these points.

2. It is unclear if the droplet property changes observed in this study are advantageous for the contained ribozyme. Is the EL R3C ladder ribozyme still active in the more viscous/solid droplets containing the longer RNAs? A ribozyme activity test started from a substrate long enough to form more viscous/solid droplets might answer this question.

Reviewer #2 (Recommendations for the authors):

The public review reflects my general view of the paper: it is an impressive achievement to get a ribozyme to control coacervate properties. The authors propose to exploit this by using the droplets to implement selection. There are still questions about the system's suitability for such a task. Readers would benefit from a clearer path forward from the authors, including an experimental assessment of a couple of important points.

A direct test of coacervate suitability for such a task would be to carry out a model in vitro selection of ligases based on their activity and influence on coacervate properties, though the system may not yet be at a stage where this is possible. In the meantime some clarity on the following points would help assess the prospects of this:

1) Coacervate enhancement of ribozyme activity: The results in Figure 1d and Figure 1b seem discordant. At the 2 h timepoint in Figure 1d, the average product length seems much lower without Lys than with – but the equivalent lanes look the same in 1b with (as far as I can tell) identical conditions. Visual inspection of the gel in 1b also suggests a much higher average product length than calculated in 1d (though perhaps this is a mirage due to staining intensity) and seen in the supporting data. If I'm missing something in my interpretation, perhaps the authors could more clearly explain the differences. This is important for readers to grasp as it underpins the claim that coacervate formation enhances ribozyme activity. I'd recommend showing a gel from the supporting figure clearly showing a condition where ligation is better with Lys. It may also be helpful to quantify the average product length in SYBR Gold stained gels (correcting for the proportionality of RNA length to signal).

2) Ribozyme activity: the observed effects of ribozyme activity on droplet properties occur when ~1/4 of the coacervate comprises ribozyme. For selection, there would be much less active ribozyme present. Can small amounts of ribozyme still influence droplet behavior when at lower concentrations (e.g. a mix of active and inactive ribozymes)?

3) Droplet fusion: There is convincing resistance of droplets to fusion when both contain active ribozyme. But in a selection setting, where a small fraction of droplets have active ribozyme and the majority do not, the relevant question is whether the droplets with inactive ribozyme will fuse with droplets with active ribozyme, i.e. does ribozyme activity in a droplet make it resistant to fusion or remove its tendency to fuse?

Reviewer #3 (Recommendations for the authors):

1 – The sentence "The increase in rate observed here is most likely due to the high concentration of ribozyme and substrate that occurs in coacervates directly formed from catalytic RNA and peptides (Ref. 15)" addresses an interesting point: How do we know that the peptide-mediated increase in ribozyme activity is due to coacervate formation as opposed to, for example, an association of freely diffusing peptides with freely diffusing ribozymes? Given that this is not the central point of the paper I would not ask for (probably difficult) experiments but for 1-2 more sentences based on reference 15 that increase the reader's understanding of how the peptide likely mediates through coacervates the increased ribozyme activity.

2 – The manuscript is of high interest in the field of origins of life, and needs to discuss in more detail the important implications of coacervates as model systems for early life forms, especially with regard to the effects of ribozyme catalysis on the material exchange between coacervate droplets. Specifically, the coupling of genotype and phenotype, the danger from molecular parasites, and the inflow/outflow of metabolites are likely affected by these changes, and this should be discussed based on the rich literature. I suggest inserting an additional paragraph into the discussion or expanding the last paragraph on these three specific points.

3 – While the longer Lys(19-72) peptides do not support ribozyme catalysis (see the first part of the results) they are used as a model system for dye exchange (Figure 3). Please state in the corresponding text the caveat that these longer peptide systems are not model systems for protocells but serve to test the effects of peptide length on dye exchange. Please also discuss whether the less pronounced effect on the exchange rates in coacervates that support ribozyme catalysis would be sufficient for evolutionary importance with respect to protocell function (see (2) above).

4 – The discussion mentions that the Lys(5-24) peptides are actually not 5-24 amino acids long but 3 to 9 amino acids in length. To avoid confusing the reader, please correct the length throughout the manuscript, and mention in the materials and methods that "commercially available (Lys)n deviates from the manufacturer's stated size range, with the (Lys)5-24 used here being predominantly comprised of oligomers between n = 3 to n = 9 in length15.".

Essential revisions:

The reviewers were generally quite positive about the work. They have made a number of suggestions that may be useful for future investigation in this area. Of these, some of these are essential as detailed below. We are not asking for any additional experiments, but please discuss the points raised by the reviewers.

1. Clarification of the title.

We agree that the previous title was somewhat ambiguous and have therefore incorporated the suggestions of reviewers 1 and 3 to retitle the paper:

Ribozyme activity modulates the physical properties of RNA-peptide coacervates

2. The results shown in figure 1b, 1d are discordant and this needs to be remedied by either choosing different images or (not as good) explaining in the text why these panels seem to be discordant.

The combination of endpoint and kinetic data is potentially confusing, and the effect of phase separation is not obvious from the SYBR Gold stained urea-PAGE gels of endpoint data. However, the use of a stained gel is necessary to visualise all of the components of the system. We have therefore attempted to clarify the data with the following measures:

• Lane profiles for relevant conditions (solution and 0.75:1 pL:RNA for Figure 1b, solution and 1.5:1 pL:RNA for Figure 1 – supplement 2) have been incorporated into the relevant figures. These profiles illustrate that the intensities of the ligated products are higher in the phase separated system, an effect which is not easily perceived in the SYBR Gold stained gel image.

• Images of the source urea-PAGE gels for the kinetic experiments (Figure 1e) have been converted into a new figure supplement (Figure 1 – supplement 3), which clearly shows the difference in rate and activity when monitoring the extension of a fluorescently-tagged substrate strand.

• We have added some brief explanation to the text to account for the discordance and to draw attention to the newly added features and figures.

We hope that these additions are sufficient to remedy the issue and discuss the issues further in our responses to reviewers 1 and 2.

3. Paragraphs 3-5 of reviewer 2's author recommendations.

Please see author recommendations for detailed responses and revisions.

Reviewer #1 (Recommendations for the authors):

1. As the products of the ligation reaction by the EL R3C ladder ribozyme are mixtures of RNAs with different lengths, the exact requirements (e.g., average/maximum length of the ligated RNAs + double strand formation) for the phenotypic changes are not clear. Is S2 or S3 enough? Are longer products (e.g., S6-S17) more important? Do the elongated RNAs need to remain hybridized with the ribozymes? Droplet formation experiments with S2 or S3 (synthesized chemically or by T7 RNAP) in the presence and absence of the inactive hybridizing ribozyme would clarify these points.

We thank the reviewer for the positive assessment of the work and helpful suggestions. Indeed, the requirements for altering material properties in these experiments are not precisely known. Follow-up studies performed in the manner suggested by the reviewer could elucidate these questions, by determining the physical properties of droplets formed from pre-reacted substrate concatemers with defined lengths or in the presence of a non- or weakly-hybridising ribozyme.

In designing our experiments, we relied on previous works (e.g. Spruijt et al. 2010) that investigated the effect of polymer length on coacervate physical properties to roughly determine initial and final RNA lengths that were likely to result in an observable change.

However, the exact conditions for achieving phenotypic change in a given system are highly specific and dependent on a broad parameter space that encompasses polymer length, RNA sequence, hybridisation state, overall polymer concentration, buffer pH, salt concentration and Mg²⁺ concentration amongst other factors. As a result, determining the exact requirements would be valuable for the further development of our model system, but would not provide general rules applicable to RNA-peptide coacervates.

2. It is unclear if the droplet property changes observed in this study are advantageous for the contained ribozyme. Is the EL R3C ladder ribozyme still active in the more viscous/solid droplets containing the longer RNAs? A ribozyme activity test started from a substrate long enough to form more viscous/solid droplets might answer this question.

The current ribozyme and substrate design did not allow us to directly address this question, as the ribozyme catalyses a pseudo-irreversible reaction that reaches an endpoint over the course of the experiment. As suggested, increasing the initial substrate length with an unstructured spacer region would be a good starting point to investigate this question.

In our previous work (Le Vay et al. 2021, https://doi.org/10.1002/anie.202109267), we reported that highly viscous or gel-like phases can occur at specific ratios in mixtures of a fragmented hairpin ribozyme and poly(L-lysine), albeit at a much lower overall polymer length. The formation of these phases was associated with increased activity, and both ribozyme activity and strand exchange were maintained, suggesting that high viscosity is not a barrier to ribozyme function in specific cases.

Reviewer #2 (Recommendations for the authors):

The public review reflects my general view of the paper: it is an impressive achievement to get a ribozyme to control coacervate properties. The authors propose to exploit this by using the droplets to implement selection. There are still questions about the system's suitability for such a task. Readers would benefit from a clearer path forward from the authors, including an experimental assessment of a couple of important points.

A direct test of coacervate suitability for such a task would be to carry out a model in vitro selection of ligases based on their activity and influence on coacervate properties, though the system may not yet be at a stage where this is possible. In the meantime some clarity on the following points would help assess the prospects of this:

1) Coacervate enhancement of ribozyme activity: The results in Figure 1d and Figure 1b seem discordant. At the 2 h timepoint in Figure 1d, the average product length seems much lower without Lys than with – but the equivalent lanes look the same in 1b with (as far as I can tell) identical conditions. Visual inspection of the gel in 1b also suggests a much higher average product length than calculated in 1d (though perhaps this is a mirage due to staining intensity) and seen in the supporting data. If I'm missing something in my interpretation, perhaps the authors could more clearly explain the differences. This is important for readers to grasp as it underpins the claim that coacervate formation enhances ribozyme activity. I'd recommend showing a gel from the supporting figure clearly showing a condition where ligation is better with Lys. It may also be helpful to quantify the average product length in SYBR Gold stained gels (correcting for the proportionality of RNA length to signal).

We thank the reviewer for the positive assessment of this work and for highlighting several possible improvements.

In response to point 1, we direct the reviewer to our response to Reviewer 1, and to the editorial team in the section “essential revisions”. In brief, the discordance stems from the use of stained gel images to illustrate the overall reaction at its endpoint in Figure 2b, but the use of a fluorescently 5’-labelled substrate strand to determine kinetics in 2d. The use of stained gels allows us to visualise all species, including the ribozyme, substrate and both linear and circular products. However, monitoring the reaction in this way complicates the determination of average substrate length due to problems of band overlap (e.g. ribozyme and S2 product) and the presence of large but low abundance circular products of unknown length, and makes visual assessment of differences in average substrate length at the endpoint of the reaction difficult.

We feel that it is important to present a stained gel which displays all reaction components and products, but have aimed to clarify the data by adding lane profiles for certain conditions, which the greater intensity of linear product bands in the presence of pL, adding a new figure supplement based on the fluorescently labelled gels from the source data of the kinetics experiment, and adding some clarification to the text.

2) Ribozyme activity: the observed effects of ribozyme activity on droplet properties occur when ~1/4 of the coacervate comprises ribozyme. For selection, there would be much less active ribozyme present. Can small amounts of ribozyme still influence droplet behavior when at lower concentrations (e.g. a mix of active and inactive ribozymes)?

This is a valid point that we feel warrants further discussion here and in the manuscript. In a more realistic scenario, it is expected that the concentrations of both functional ribozyme and compatible substrate would be much lower, and that the remaining material would contain non-functional (or at least non-ligating) RNA strands and perhaps other charged polymers.

It is therefore necessary to improve the catalytic properties of the ribozyme in order to move towards a more realistic system. In Figure 1 – supplement 1, we show that increasing the amount of substrate relative to the ribozyme actually leads to a reduction in ribozyme activity. We therefore anticipate that optimising the ribozyme for multi-turnover reaction might be required to achieve significant phenotypic change in a system with small amounts of ribozyme but abundant substrate. In our previous work, we demonstrated that the Hairpin ribozyme is capable of strand exchange and therefore multiple substrate ligations in RNA-peptide condensates, perhaps due to the short length of the substrate binding strand. By altering the substrate binding arms of the R3C ribozyme, it might be possible to engineer similar behaviour, perhaps at the cost of reaction rate.

In more complex mixtures with a greater variety of RNA strands and therefore a less well-defined substrate pool, a ribozyme with less stringent substrate sequence requirements might be better suited to alter material properties. The sunY ribozyme, which requires only a 5’-G for substrate ligation, would be an interesting candidate for this.

We have modified the final paragraph of the discussion to account for some of these shortcomings.

3) Droplet fusion: There is convincing resistance of droplets to fusion when both contain active ribozyme. But in a selection setting, where a small fraction of droplets have active ribozyme and the majority do not, the relevant question is whether the droplets with inactive ribozyme will fuse with droplets with active ribozyme, i.e. does ribozyme activity in a droplet make it resistant to fusion or remove its tendency to fuse?

This is a highly relevant but experimentally challenging question, which we are currently attempting to address as part of our ongoing projects. The orthogonally labelled substrates transfer between droplet populations by both fusion and diffusion, and as a result all droplets contain measurable amounts of both fluorophores shortly after mixing. This means we are currently unable to investigate the behaviour of each droplet population independently to determine if e.g. an active population resists fusion when mixed with an inactive population. Our preliminary results suggest that in mixtures of active and inactive droplets, the labelled RNA from the active population remains highly localised, whilst the labelled RNA from the inactive population diffuses evenly throughout both populations. Further work is required to investigate fusion in these systems, in particular to establish methods of orthogonally measuring droplet size in the two populations.

Reviewer #3 (Recommendations for the authors):

1 – The sentence "The increase in rate observed here is most likely due to the high concentration of ribozyme and substrate that occurs in coacervates directly formed from catalytic RNA and peptides (Ref. 15)" addresses an interesting point: How do we know that the peptide-mediated increase in ribozyme activity is due to coacervate formation as opposed to, for example, an association of freely diffusing peptides with freely diffusing ribozymes? Given that this is not the central point of the paper I would not ask for (probably difficult) experiments but for 1-2 more sentences based on reference 15 that increase the reader's understanding of how the peptide likely mediates through coacervates the increased ribozyme activity.

We would like to thank the reviewer for this point, and agree that the claim was not fully supported. In response to this and a similar point raised by Reviewer 1, we have performed additional experiments investigating the activity of the ribozyme at varying PEG, Mg²⁺ and RNA concentrations. These experiments reveal that both high RNA and Mg²⁺ concentrations are required for enhanced activity. We direct the reviewer to the earlier response to Reviewer 1, and to the new data presented in Figure 1 – supplement 4.

2 – The manuscript is of high interest in the field of origins of life, and needs to discuss in more detail the important implications of coacervates as model systems for early life forms, especially with regard to the effects of ribozyme catalysis on the material exchange between coacervate droplets. Specifically, the coupling of genotype and phenotype, the danger from molecular parasites, and the inflow/outflow of metabolites are likely affected by these changes, and this should be discussed based on the rich literature. I suggest inserting an additional paragraph into the discussion or expanding the last paragraph on these three specific points.

We agree that the ribozyme induced changes to coacervate properties may impact factors relevant to early compartmentalised replicators such as parasite resistance. However, as the ribozyme system described in this work is not replicative, we would like to limit the discussion of these aspects to avoid lengthening the manuscript further. We have included a sentence in the discussion highlighting these areas as interesting avenues for further study.

3 – While the longer Lys(19-72) peptides do not support ribozyme catalysis (see the first part of the results) they are used as a model system for dye exchange (Figure 3). Please state in the corresponding text the caveat that these longer peptide systems are not model systems for protocells but serve to test the effects of peptide length on dye exchange. Please also discuss whether the less pronounced effect on the exchange rates in coacervates that support ribozyme catalysis would be sufficient for evolutionary importance with respect to protocell function (see (2) above).

We thank the reviewer for bringing this miscommunication to light. The longer lysine polymers do indeed inhibit ribozyme activity, but only at ratios when the lysine oligopeptides are in excess over RNA i.e. (Lys)_19-72: RNA > 1:1, as determined by the lysine titration PAGE in Figure 1b. In subsequent experiments, we selected a (Lys)_19-72: RNA ratio of 0.75:1 at which the compartmentalised ribozymes are highly active and coacervates form in liquid state. We believe that under these conditions the droplets containing longer peptides are an interesting model system. In addition, all experiments are also performed with droplets formed from the shorter peptide and the difference in behaviour between these systems is discussed. This point has been clarified in the second paragraph of the Results section.

4 – The discussion mentions that the Lys(5-24) peptides are actually not 5-24 amino acids long but 3 to 9 amino acids in length. To avoid confusing the reader, please correct the length throughout the manuscript, and mention in the materials and methods that "commercially available (Lys)n deviates from the manufacturer's stated size range, with the (Lys)5-24 used here being predominantly comprised of oligomers between n = 3 to n = 9 in length15.".

We thank the reviewer for bringing this miscommunication to light. The longer lysine polymers do indeed inhibit ribozyme activity, but only at ratios when the lysine oligopeptides are in excess over RNA i.e. (Lys)_19-72: RNA > 1:1, as determined by the lysine titration PAGE in Figure 1b. In subsequent experiments, we selected a (Lys)_19-72: RNA ratio of 0.75:1 at which the compartmentalised ribozymes are highly active and coacervates form in liquid state. We believe that under these conditions the droplets containing longer peptides are an interesting model system. In addition, all experiments are also performed with droplets formed from the shorter peptide and the difference in behaviour between these systems is discussed. This point has been clarified in the second paragraph of the Results section.

Author details

1. Kristian Kyle Le Vay

   Department of Chemistry and Chemical Biology, TU Dortmund University, Dortmund, Germany

   Contribution

   Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Investigation, Visualization, Methodology, Writing – original draft, Project administration, Writing – review and editing

   Contributed equally with

   Elia Salibi

   For correspondence

   kristian.levay@tu-dortmund.de

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2455-8706

2. Elia Salibi

   Department of Chemistry and Chemical Biology, TU Dortmund University, Dortmund, Germany

   Contribution

   Formal analysis, Investigation, Visualization, Methodology, Writing – original draft, Writing – review and editing

   Contributed equally with

   Kristian Kyle Le Vay

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0009-0003-8201-4237

3. Basusree Ghosh

   Max-Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany

   Contribution

   Investigation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

4. TY Dora Tang

   Max-Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany

   Contribution

   Conceptualization, Resources, Supervision, Funding acquisition, Writing – review and editing

   For correspondence

   tang@mpi-cbg.de

   Competing interests

   No competing interests declared

5. Hannes Mutschler

   Department of Chemistry and Chemical Biology, TU Dortmund University, Dortmund, Germany

   Contribution

   Conceptualization, Resources, Supervision, Funding acquisition, Methodology, Project administration, Writing – review and editing, Writing – original draft

   For correspondence

   hannes.mutschler@tu-dortmund.de

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8005-1657

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