Oligoarginine peptides slow strand annealing and assist non-enzymatic RNA replication.

The non-enzymatic replication of RNA is thought to have been a critical process required for the origin of life. One unsolved difficulty with non-enzymatic RNA replication is that template-directed copying of RNA results in a double-stranded product. After strand separation, rapid strand reannealing outcompetes slow non-enzymatic template copying, which renders multiple rounds of RNA replication impossible. Here we show that oligoarginine peptides slow the annealing of complementary oligoribonucleotides by up to several thousand-fold; however, short primers and activated monomers can still bind to template strands, and template-directed primer extension can still occur, all within a phase-separated condensed state, or coacervate. Furthermore, we show that within this phase, partial template copying occurs even in the presence of full-length complementary strands. This method to enable further rounds of replication suggests one mechanism by which short non-coded peptides could have enhanced early cellular fitness, and potentially explains how longer coded peptides, that is, proteins, came to prominence in modern biology.

RNA has been postulated to be the biopolymer from which early life on Earth evolved, due to the central role of RNA as a mediator of information transfer between DNA and proteins, and to the ability of RNA to act as both a propagator of genetic information and as a catalyst. Most notably, RNA is the catalyst responsible for the ribosomal synthesis of all coded proteins[1-3], strongly suggesting that RNA-based catalysis preceded the evolution of coded peptide synthesis. Furthermore, recent findings point to a potential prebiotic pathway for the synthesis of ribonucleotides and thus RNA[4-6]. If RNA was indeed the original biopolymer of cellular life, then selective pressures for faster and more accurate RNA replication would likely have led to the evolution of an RNA polymerase ribozyme that could catalyze the replication of increasingly complex RNA genomes. However, prior to the evolution of the first RNA polymerase ribozyme, RNA must have replicated nonenzymatically[3,7]. This conclusion has motivated a long history of efforts to copy RNA templates nonenzymatically, and although the efficient copying of arbitrary template sequences has not yet been demonstrated, recent advances[7-11] suggest that such template copying may well be possible.

Given the potential for nonenzymatic template copying to generate seminal RNA strands, one must then ask what additional steps are required to enable repeated cycles of RNA replication. The nonenzymatic copying of a template strand results in the formation of an RNA duplex, which must then denature to provide templates for the next round of replication. We have previously shown that the thermal separation of the strands of an RNA a duplex is facilitated by the incorporation of a fraction of 2′–5′ linkages in the RNA backbone; these linkages form as a consequence of nonenzymatic template copying and significantly lower the melting temperature of the resulting duplex[12]. However, subsequent rounds of nonenzymatic RNA replication are inhibited by the rapid reannealing of the separated strands following heating and cooling[13], preventing the weakly-binding RNA primers and activated monomers required for polymerization from associating with the template[14] (Fig. 1a). In order for subsequent rounds of replication to be possible, reannealing of the separated single strands must occur on a time scale that is comparable to or slower than the rate of strand copying. In principle, this kinetic control could be accomplished by operating in a highly dilute regime (≤1 nM RNA)[7,13]; however, 1 nM RNA corresponds to only a few strands per protocell 3–4 μm in diameter. Protocells containing only a few strands of RNA would not have contained a sufficient concentration of an RNA with catalytic activity to confer a benefit to the protocell[7]. We therefore sought to identify conditions under which RNA strand reannealing at more relevant μM concentrations is significantly slowed, while minimally affecting template-copying chemistry.

Figure 1

The reannealing problem and a proposed solution. a) Complete template-directed primer extension results in a full-length duplex (newly synthesized strand in maroon, original template in gray) (1). After strand separation by heating (2), subsequent cooling results in rapid reannealing of the newly synthesized complementary strand to the template strand (1); this prevents primer-template binding, outcompeting the slow process of nonenzymatic RNA polymerization (3) thereby preventing further rounds of RNA replication. b) RNA-binding oligoarginine peptides (green) inhibit strand annealing and promote further rounds of nonenzymatic replication. After an RNA duplex is formed (1), the strands are separated by heating (2). Subsequent cooling allows the peptide to bind to the separated complementary strands but not to the shorter RNA primers. This selectivity prevents reannealing of the full-length replicated strands, allowing each strand to act as a template, to which shorter primers can then bind (3). The nonenzymatic polymerization reaction is free to proceed, resulting in a complete replication cycle that would not be possible without the peptide.

Considerable evidence supports the possibility that peptides and RNA could have been present together[15-17] on the primitive earth. Atmospheric discharge experiments[18,19], transport from meteors and cosmic dust[20,21], and more recent scenarios for prebiotic amino acid synthesis[16,17] all point towards the existence of amino acids on the earth’s surface shortly after its formation. Although early studies indicated that the basic amino acids arginine (Arg, R) and lysine (Lys, K) would have been among the least abundant[22,23], recent studies illustrate a prebiotically plausible arginine synthesis from simple precursors in a cyanide-rich reducing environment in the presence of hydrogen sulfide[24]. Arginine is particularly interesting functionally, since arginine-rich oligopeptides are known to bind strongly to both DNA and RNA[25,26]; the DNA condensing protein protamine is mostly composed of arginines[27,28] and Arg3 has been reported to condense DNA in vitro via electrostatic interactions[29]. If arginine-rich peptides could have assembled on the early Earth, it seems likely that they would have interacted strongly with RNA, potentially enhancing or inhibiting aspects of RNA replication and function. A variety of prebiotically plausible mechanisms for peptide bond formation have also been explored[30-32] including the solution-phase elongation of the peptide chain by the stepwise addition of either N-carboxyanhydrides (NCA) or 2-thiono-5-oxazolidones, formed by the reaction of amino acids with the volcanic gases carbonyl sulfide[33] or carbon disulfide[34], respectively. Although most of these processes lack sequence specificity, efficient oligoarginine production via polymerization of the L-arginine-NCA has been reported through a unique mechanism that involves a six-membered ring intermediate[35]. The polymerization of arginine on acidic mineral surfaces provides an alternative experimentally demonstrated pathway to oligoarginine peptides that could operate in the presence of a complex mixture of amino acids[36].

Given the potential existence of arginine-rich peptides on the early Earth, coupled with the ability of these peptides to bind to RNA, we sought to explore the nature and consequences of such RNA-peptide interactions on nonenzymatic replication. In the course of these investigations, we discovered that short arginine-rich peptides can prevent the annealing of complementary RNA strands in a concentration- and length-dependent manner. In the present work, we simulate a post-replication round of nonenzymatic RNA polymerization by thermally denaturing an RNA duplex, and show that template-directed primer extension proceeds only in the presence of an oligoarginine peptide (Fig. 1b). We also show that the nonenzymatic primer extension reaction occurs within a phase-separated condensed state, i.e. a coacervate, formed by the electrostatic binding of oppositely charged RNA and oligoarginine polyelectrolytes. Taken together, these results show that cationic peptides could have enhanced the fitness of an emerging protocell by assisting in multiple rounds of replication.

Results

Binding of RNA to Oligoarginine Peptides

As a prelude to exploring the functional consequences of the binding of arginine-rich peptides to RNA oligonucleotides, we used a series of independent assays to examine the binding of oligoarginine peptides (R10NH2 and R9NH2, NH2:C-terminal amide) to short RNA oligonucleotides. Initial gel electrophoresis assays showed that the oligoarginine peptide R10NH2 at concentrations ≥100 μM caused a visible shift in the mobility of an RNA 15-mer (Supplementary Fig. S1). Furthermore, the fluorescence emission intensity of a 2-aminopurine (2AP)-containing RNA 15-mer increased significantly upon addition of 100 μM R9NH2 (Supplementary Fig. S2). As 2AP fluorescence is quenched by the stacking of adjacent bases[37], the increase in fluorescence suggests that peptide binding alters the conformation of ssRNA such that base-stacking is decreased. Finally, a solution containing a fluorescently labeled RNA 15-mer became turbid in the presence of 10 mM R9NH2 due to the formation of aggregated peptide-RNA complexes (Supplementary Fig. S3). The driving force for this complexation is most likely electrostatic as seen previously for the association of polylysine with DNA[38].

In order to examine the effect of peptide and RNA length on the concentration dependence of the interaction, we turned to fluorescence anisotropy titrations. We observed that the fluorescence anisotropy of a 2AP-containing RNA 15-mer increased with increasing peptide concentrations; an increase in fluorescence anisotropy indicates a decrease in the rate of rotational diffusion, consistent with peptide binding to RNA (Fig. 2a). The presence of R10NH2 resulted in a greater increase in the fluorescence anisotropy compared to R7 and R5 at the same peptide concentrations; addition of R5 did not result in any significant increase in the fluorescence anisotropy. Likewise, addition of R10NH2 to an RNA 15-mer resulted in a greater increase in the fluorescence anisotropy compared to addition of R10NH2 to a 10-mer, while R10NH2 addition to a 7-mer did not result in any significant increase in the fluorescence anisotropy (Fig. 2b). These data indicate that longer peptides and RNA bind more tightly to each other than do their shorter counterparts, a result which would be expected from association driven by electrostatic interactions[39].

Figure 2

RNA-peptide binding measured by fluorescence anisotropy and circular dichroism. Experiments were performed in annealing buffer (Methods). Error bars indicate ± one SEM. a) Fluorescence anisotropy of a 2-aminopurine (2AP)-containing RNA 15-mer (5′-CC(2AP)GUCAGUCUACGC-3′, 10 μM) in the presence of three oligoarginine peptides of different lengths (R5, R7, and R10NH2; NH2: C-terminal amide) over increasing peptide concentrations. Higher peptide concentrations and greater peptide length lead to increased fluorescence anisotropy. b) Fluorescence anisotropy of 2AP-containing RNAs of increasing lengths (7-mer, 10-mer, and 15-mer, 10 μM; see Methods for sequences) with increasing R10NH2 concentrations. Longer RNAs show a greater increase in anisotropy. c) Circular dichroism (CD) traces of an RNA 15-mer (5′-CCAGUCAGUCUACGC-3′, 5 μM) with increasing concentrations of R10NH2. The initial spectrum is characteristic of an A-type helical conformation[40]. The molar circular dichroism (Δε) of the 270 nm peak decreases with increasing peptide concentration (up to 25 μM).

To further explore the possibility of a conformational change in the RNA upon peptide binding, we monitored the circular dichroism (CD) spectrum of a single-stranded RNA 15-mer in the presence of increasing concentrations of R10NH2 (Fig. 2c). The spectrum in the absence of peptide is characteristic of an A-type helical conformation[40], but the 270 nm peak decreases in intensity with increasing peptide concentration, indicating that the global helical structure of the single-stranded RNA is significantly disrupted upon peptide binding. Finally, we measured the effect of addition of Mg2+ to a pre-formed RNA-peptide complex. Both fluorescence anisotropy (Supplementary Fig. S4) and CD (Supplementary Fig. S5) measurements show that added Mg2+ displaces the peptide in the RNA-peptide complex, presumably by disrupting the electrostatic interactions between the peptide and RNA.

Oligoarginine Peptides Interfere with RNA Annealing

The formation of charge-neutralized RNA-peptide complexes suggested that peptide binding might increase the rate of annealing of complementary strands; on the other hand, the apparent change in RNA conformation upon peptide binding hinted that the rate of annealing might be decreased. To address these hypotheses we measured the effect of oligoarginine peptides on the second-order rate of annealing of complementary RNA 15-mers by stopped-flow fluorometry (Fig. 3a). Each RNA strand was separately incubated with peptide at room temperature for at least one hour to ensure maximum peptide-RNA binding before annealing. A single 2AP residue present in one RNA strand was employed as a reporter of the fraction of RNA in duplex form, as 2AP fluorescence is quenched upon duplex formation[37]. RNA melting analysis showed that replacing one A residue in one of the strands with 2AP does not compromise duplex stability at room temperature (Supplementary Fig. S6). We observed that the t1/2 (initial second-order annealing half-life) values for two RNA 15-mers increased from 0.9(1) s in the absence of peptides, up to a maximum of 5.2(5) × 103 s (SEM values in parentheses) in the limit of increasing concentrations and lengths of oligoarginines (Fig. 3b); for the most effective peptide, R10NH2, no further inhibition of annealing was observed above a concentration of ~15 μM (see Supplementary Table S1 for the annealing t1/2 for all conditions tested), perhaps due to the formation of a phase-separated coacervate at concentrations >15 μM R10NH2 (Supplementary Fig. S7). To confirm that this annealing inhibition was due to the cationic nature of the oligoarginine peptides, we tested the anionic peptide GDGEGDGEGD (G: glycine, D: aspartate, E: glutamate), which did not result in a significant increase in the RNA annealing t1/2 (Supplementary Fig. S8).

Figure 3

RNA annealing rates in the presence of peptides. Initial second-order annealing half-lives (t1/2) were obtained for an RNA 15-mer (5′-GCGUAGACUGACUGG-3′) and its 2AP-containing complement (5′-CC(2AP)GUCAGUCUACGC-3′) in annealing buffer (Methods) at RNA concentrations of 1 μM (See Supplementary Methods for fitting parameters and Supplementary Table S1 for a list of all conditions tested). Error bars indicate ± one SEM. a) Kinetic traces showing the annealing of the two RNA 15-mers in the presence or absence of 100 μM R5, R7, R9, or R10NH2. b) t1/2 for RNA 15-mers with increasing concentrations of R5, R7, R9, and R10NH2. c) t1/2 for RNA of different lengths (7-mers to 15-mers, on x-axis; see Supplementary Methods for sequences) annealing to the 2AP-containing 15-mer with or without 100 μM R10NH2. Longer RNAs exhibit slower annealing kinetics in the presence of peptide.

As the fluorescence anisotropy results (Fig. 2b) indicate that R10NH2 binds to longer RNAs more strongly than shorter RNAs, we investigated whether R10NH2 could selectively inhibit the annealing of longer RNAs. Indeed, we observed that after R10NH2 addition the annealing rate of RNA decreased more for longer RNA oligonucleotides (Fig. 3c). Remarkably, a 15-mer anneals roughly two orders of magnitude more rapidly to a 7-mer (t1/2 = 71(7) s) than to its full-length complementary strand (t1/2 = 5.2(5) × 103 s) in the presence of 100 μM R10NH2. We then asked whether the ions and ionic compounds required for nonenzymatic replication[9] would reduce the degree of peptide-conferred annealing inhibition. In the presence of 100 μM R10NH2, the addition of Mg2+ (up to 50 mM, Supplementary Fig. S9), the activated monomer cytidine 5′-phosphor-2-methylimidazolide (2-MeImpC, up to 50 mM, Supplementary Fig. S10), cytidine monophosphate (up to 50 mM, Supplementary Fig. S11), and sodium citrate (up to 25 mM, Supplementary Fig. S12) all significantly increased the annealing rate of two RNA 15-mers (by up to two orders of magnitude). High concentrations of Tris-Cl buffer (up to 500 mM) also increased the annealing rate of two RNA 15-mers in the presence of R10NH2 but to a lesser extent (Supplementary Fig. S13). These observations are consistent with the binding of oligoarginine peptides to RNA by electrostatic interactions, which are disrupted by monovalent and divalent cations. Finally, we also confirmed that the ability of R10NH2 to slow RNA annealing is not significantly affected by pH within the range of pH 6–9 (Supplementary Fig. S14). We suggest that the binding of oligoarginine to RNA changes the RNA structure significantly and contributes to annealing inhibition. At sufficiently high concentrations, added oligoarginine results in the formation of a condensed phase (Supplementary Fig. S7) driven by charge-neutralization, not unlike those observed for ATP in the presence of polyvalent cations[41,42].

Oligoarginine-Assisted Nonenzymatic RNA Replication

The observation that R10NH2 greatly slows the annealing of two RNA 15-mers while having little effect on the rate of annealing of a 7-mer to a 15-mer led us to hypothesize that in the presence of R10NH2 a short primer would still be able to anneal to a longer template even in the presence of the complementary strand to the template, allowing primer extension to occur. Traditionally, high concentrations (≥50 mM) of Mg2+ and activated monomer are used in nonenzymatic replication experiments[9]. However, because both Mg2+ and activated monomers disrupt the electrostatic interactions between R10NH2 and RNA, we performed all nonenzymatic polymerization studies at lower concentrations of Mg2+ and monomer (10 mM each). The annealing of complementary 15-mers in buffer conditions simulating a nonenzymatic primer extension experiment (10 mM MgCl2, 10 mM 2-MeImpC, and 250 mM Na-HEPES pH 8) is still effectively slowed by 100 μM R10NH2 (t1/2 = 8.5(4) × 102 s). Because R10NH2 alters the conformation of ssRNA, we then asked whether R10NH2 would inhibit nonenzymatic primer extension by measuring the rate of primer extension on a 16-mer C4 template using guanosine 5′-phosphor-2-methylimidazolide (2-MeImpG). We found that the rate of primer extension decreased by only ~30% in the presence of 1 mM of the R10NH2 peptide (Figs. 4a–b and Supplementary Fig. S15)—in contrast to the greater than three order-of-magnitude increase in the annealing t1/2 of two RNA 15-mers in the presence of 100 μM R10NH2 (Fig. 3). To our satisfaction, a promising result was obtained when the complementary strand to a template, preincubated with R10NH2, was added to a preformed primer-template complex, also preincubated with peptide (Fig. 4c). Under these conditions, it appears that the peptide effectively inhibited the annealing of the complementary strand to the template, while still allowing the primer extension reaction to proceed, whereas in the absence of peptide, the addition of the complementary strand resulted in complete inhibition of the primer-extension reaction.

Figure 4

Nonenzymatic RNA polymerization. Reactions performed with 10 mM MgCl2, 250 mM Na-HEPES pH 8, and 10 mM 2-MeImpG. a) Polyacrylamide gel of nonenzymatic additions of 2-MeImpG to a 5′-cyanine 3 (Cy3)-labeled primer (5′-Cy3-CAGACUGG-3′, 2 μM) on a C4 template (5′-AACCCCCCAGUCAGUC-3′, 2.5 μM) with or without 1 mM R10NH2. Bolded cytosines represent 2-MeImpG binding sites on the template. b) log of the fraction of unreacted primer for gel lanes in a vs. time, with (red) or without (black) R10NH2. The slope of the lines (linear fit, R2 = 0.99) represents the pseudo-first-order rate constant, kobs, in h−1 for the respective reactions. With 1 mM R10NH2, kobs = 0.084(5) h−1. With no peptide, kobs = 0.110(5) h−1. SEM in parentheses. n = 5. c) Polyacrylamide gel of an overnight nonenzymatic primer extension experiment with 1.2 μM primer and 1.25 μM template (incubated with or without 1.5 mM R10NH2), after addition of 0 μM or 2 μM complementary strand to the template (5′-GACUGACUGGGGGGUU-3′) separately incubated with or without 1.5 mM R10NH2. d) Post-replication round of nonenzymatic primer extension. The template (gray) and its complement (red) were annealed, then peptide, monomer, and primer (blue) were added and the mixture was heated briefly to 95 °C. After cooling to 4 °C to allow primer-template binding, the system was allowed to warm to room temperature. e) Polyacrylamide gel of the overnight primer extension experiment described in d with 0.95 μM primer, 1 μM template, and with or without 1.2 μM complementary strand and 1.5 mM R10NH2, respectively.

We then turned our attention to simulating a post-replication round of nonenzymatic template-directed primer extension by starting with an RNA duplex, i.e. a template strand already bound to its complement. We added the other components of the nonenzymatic primer extension reaction (primer, Mg2+, and 2-MeImpG) and briefly heated the sample to 95 °C to melt the duplex followed immediately by cooling on ice. Rapid cooling is necessary (Supplementary Fig. S16), most likely to allow peptide-RNA binding to occur before the annealing of complementary RNA strands. The primer extension reaction was then allowed to proceed as the reaction mixture warmed to room temperature overnight (Fig. 4d). In the absence of the oligoarginine peptide, no primer extension was observed, as expected from rapid annealing of the complementary strand to the template. In the presence of peptide, however, the primer extension reaction did proceed (Fig. 4e); we propose that the thermally separated template and complement each tightly binds to the peptide, which prevents them from reannealing. However, the shorter primer binds more weakly to the peptide and is still able to hybridize to the template, thus allowing nonenzymatic primer extension to proceed under conditions approximating a post-replication round of RNA polymerization.

Primer extension within the RNA-Peptide Condensed Phase

As we observed the formation of phase-separated droplets when R10NH2 was added to both single-stranded (Fig. 5a) and duplex RNA (Fig. 5b), we suspected that the primer extension reaction was occurring in the highly concentrated RNA-peptide coacervate rather than in the very dilute aqueous phase. To begin to test this hypothesis, we used confocal fluorescence microscopy to monitor the localization of a 5′-cyanine 3 (Cy3)-labeled RNA 8-mer primer in the presence of R10NH2 and a complementary RNA 15-mer. As expected, the 8-mer and 15-mer phase-separate together into large (~5–10 μm) globular structures (Fig. 5c); this phase separation phenomenon is dependent on a variety of factors including the presence of peptide and a complementary RNA (Supplementary Fig. S17), the salt concentration (Supplementary Figs. S18–S19), and the length of the RNA itself (Supplementary Figs. S20–S21). A quantitative UV spectroscopic analysis also revealed that in the presence of R10NH2 and a complementary 15-mer, roughly 97% of a native RNA 7-mer resides in the condensed phase (Supplementary Figs. S22–S23, Supplementary Table S2). These observations suggest that the primer extension reaction occurs within the coacervate phase, since that is where most of the primer is localized.

Figure 5

Condensed phase of RNA. Scale bars, 10 microns. a) Confocal fluorescence microscopy image (488 nm excitation, 525 nm emission) of a 5′-6-carboxyfluorescein (FAM)-labeled RNA 15-mer (5′-FAM-CCAGUCAGUCUACGC-3′, 5 μM) with 1 mM R10NH2 in annealing buffer (Methods). b) Image of the sample from a but with an added complementary RNA 15-mer (5′-GCGUAGACUGACUGG-3′, 5 μM). c) Confocal fluorescence microscopy image (561 nm excitation, 595 nm emission) of a 5′-Cy3-labeled RNA 8-mer (5′-Cy3-CAGACUGG-3′, 10 μM) with a complementary RNA 15-mer (5′-CCAGUCAGUCUACGC-3′, 10 μM) with 1 mM R10NH2, 100 mM Tris-Cl pH 8, and 10 mM MgCl2. d) Nonenzymatic primer extension experiment after 4 hours with the Cy3-labeled RNA primer (1.25 μM) and template (1.4 μM) from Fig. 4, 10 mM MeImpG, 10 mM MgCl2, 250 mM Na-HEPES pH 8, and with or without 1 mM R10NH2. A sample containing peptide was immediately centrifuged and the supernatant was isolated from the condensed-phase pellet; the reaction was then allowed to proceed separately in the supernatant and in the pellet.

To show directly that the observed primer extension reaction was occurring in the coacervate droplets, we centrifuged a solution containing peptide and all components of a nonenzymatic primer extension experiment and separated the supernatant from the pellet containing the coacervate phase. We then allowed the primer extension reaction to proceed separately in both the supernatant and the coacervate phase. Subsequent analysis of the reaction products confirmed that in the presence of peptide, a majority of the primer extension reaction occurred within the isolated coacervate phase (Fig. 5d). We suggest that in the presence of peptide, shorter RNA strands, i.e. primers, that bind weakly to peptide are still able to diffuse freely, and hence are still able to hybridize to the template strands in the condensed phase and undergo polymerization.

Discussion

One of the seldom-addressed problems with the RNA world hypothesis is that in order for multiple generations of nonenzymatic RNA replication to occur, the new single-stranded templates generated by melting the duplex product of template copying must reanneal on a time scale comparable to or slower than the time scale of template copying. In practice, this is a formidable challenge, since the reannealing of complementary RNA strands at reasonable concentrations (~1 μM) is extremely fast (t1/2 ≈ 1 s), while RNA copying chemistry, at least in the current state of the art, is quite slow, occurring over hours to days[10]. We have demonstrated here that the binding of oligoarginine peptides to complementary strands of RNA selectively slows down strand reannealing by up to several thousand-fold. This kinetic property allows template-directed nonenzymatic RNA polymerization to occur in the presence of a full-length strand complementary to the template (Fig. 4e). Surprisingly this nonenzymatic primer extension reaction occurs in a condensed coacervate phase formed as a consequence of the electrostatic association of the oligoarginine peptides with RNA oligonucleotides (Fig. 5). Within the coacervate phase the annealing of longer RNA oligomers is selectively inhibited (Fig. 3), thus allowing time for primer extension to occur before strand annealing is complete. Further exploration and optimization of the selective inhibition of strand annealing has the potential to allow multiple rounds of RNA replication to occur.

In light of previous research showing the potential plausibility of prebiotic arginine synthesis and peptide bond formation[30], the possibility that arginine-rich peptides facilitated continued cycles of RNA replication in a prebiotic setting should be considered. We note that the prebiotic synthesis of arginine-rich peptides would be enhanced by the existence of local environments whose ambient chemistry favored arginine synthesis[24]; another potentially important factor is that the oligomerization of arginine specifically, even in the presence of other amino acids, could be templated by negatively charged surfaces such as minerals[36], fatty acid membranes, or even RNA itself[43]. Assuming the simultaneous existence of arginine-rich peptides and RNA on the early earth, a primitive cell would gain a considerable evolutionary advantage if it were able to internally synthesize arginine-rich peptides, perhaps foreshadowing the evolution of coded translation and the later appearance of the arginine-rich DNA condensing proteins such as protamine[27,28] and the histones[44] found in modern cells. However, the concentrations of oligoarginine peptides used in this study for successful nonenzymatic template-directed primer extension in the presence of a competing complementary strand are quite high (1.5 mM) and it is unclear how such concentrations could be reached in a prebiotic system or whether lower concentrations of peptide could have had the same effect. As it is known that nucleic acids electrostatically bind to many cationic polymers[45], the inhibition of RNA annealing could very well have been a result of the strong binding of RNA to any cationic polymer, some of which may be more effective than oligoarginine. Thus, we cannot discount the possibility that a presently unknown cationic prebiotic polymer, potentially with a simpler structure and/or synthesis than oligoarginine, e.g. a polyamine[42,46], could also efficiently slow annealing. In addition, the recent discovery of complex macrostructures resulting from self-assembling cationic tripeptides[47] suggests that the supramolecular assembly of simple prebiotic peptides to form a cationic RNA-binding matrix should also be considered as a future avenue of investigation.

Although we have shown that oligoarginine binding to RNA slows complement-template annealing, the mechanism by which this occurs remains to be elucidated. In principle, the relevant RNA-peptide interaction could be either thermodynamically or kinetically controlled. In the thermodynamically controlled extreme, the addition of the peptide to double-stranded RNA would destabilize the duplex, forming peptide-single-stranded RNA complexes over time. In this scenario, binding of oligoarginine to a single strand of RNA must be much stronger than binding to the duplex, so that the peptide would cause the RNA duplex to dissociate. A short primer, being less strongly bound to peptide than the full-length RNA[39], could then hybridize to the template-peptide complex and subsequently take part in template-directed primer extension. However, we do not observe primer extension products without a heating and immediate cooling step (Supplementary Fig. S16), which strongly argues for a kinetically controlled mechanism in which the reannealing of longer strands is selectively inhibited after strand separation. This retardation of hybridization could occur if, for example, peptide binding causes a conformational change in the RNA that prevents the nucleation of base-pairing required for efficient annealing[48,49]. As we have shown, the peptide binds only weakly to short primer strands, and this fact allows the primer to bind to the template, thus permitting the primer extension reaction to proceed. We also propose that a rapid cooling step immediately after strand separation is necessary as it promotes peptide-RNA binding on a timescale competitive with the reannealing of two longer strands. Thus the prebiotic accessibility of a steep temperature gradient is critical for successful replication in the system we studied. Thermal convection and thermophoresis within the porous rocks of hydrothermal vents[50] and hydrothermal circulation generated by hydrothermal vents in ponds or lakes[51] are two prebiotically plausible environments that would allow biomolecules to access both very hot and very cold aqueous environments on a timescale fast enough to promote successful nonenzymatic replication.

In a search for further evidence that would distinguish between thermodynamically and kinetically controlled mechanisms, we consider the differences between the CD spectra of single- versus double-stranded RNA 15-mers upon addition of R10NH2. Single-stranded RNA in the absence of peptide exists predominantly in an A-form helical conformation, but this conformation is disrupted after binding to R10NH2 as evidenced by the disappearance of the 270 nm peak in the CD spectrum (Supplementary Fig. S5). In contrast the 270 nm peak in the CD spectrum of duplex RNA does not disappear after addition of R10NH2 (Supplementary Fig. S24); disappearance of the 270 nm peak would have indicated possible destabilization and/or denaturation of the duplex by peptide binding. Instead, the 270 nm peak shifts to a maximum at 310 nm, consistent with a conformational change. This spectral data provides evidence that the strands of the 15-mer RNA duplex do not separate upon addition of R10NH2, supporting a mechanism of peptide-mediated primer extension that is not thermodynamically controlled. In fact, the CD spectrum may suggest specific binding between an RNA duplex and R10NH2; further investigations into the nature of this complex are ongoing.

It has recently been shown that a variety of phase-separated systems can be encapsulated inside model protocells[41]. The assembly of a fatty acid vesicle around an oligolysine-RNA coacervate has also been reported[52], which is significant as lysine oligomers are also able to slow strand annealing, albeit less effectively than oligoarginine (Supplementary Fig. S25). These results, along with the observations that arginine-rich peptides can localize RNA to[53] and are compatible with (Supplementary Fig. S26) fatty acid membranes, suggest that oligoarginine-RNA coacervates may also be encapsulated within fatty acid-based model protocell membranes. The encapsulation of coacervate compartments within a model protocell is particularly interesting as it could have been a mechanism by which primitive cells organized and concentrated compounds and functionality, perhaps resulting in the earliest organelles[42,54]. Remarkably, modern eukaryotic cells also utilize phase-separated RNA-protein granules for a variety of cellular functions, including segregation and concentration of RNA and proteins[55-57]. It is unknown whether these modern coacervates are a relatively recent innovation, or reflect an ancient evolutionary origin.

In order to better understand the origin of life, we aim to design and construct a model protocell system[9]—for example a fatty acid vesicle containing templates, primers, activated mononucleotides, peptides, and catalytic metal ions—in which multiple rounds of nonenzymatic primer extension reactions can be induced by thermal cycling and can be iterated ad infinitum. Multiple rounds of replication would bring us one step closer to emulating the first primitive cells that were able to grow, divide, and evolve under early earth conditions.

Methods

Peptides, RNA, and Nucleotides

Peptides (R5, R7, R9, R9NH2, and R10NH2; NH2: C-terminal amide) were purchased from either NeoBioLab (Woburn, MA) or GenScript (Piscataway, NJ) at >95% purity as trifluoroacetic acid salts. DNA and RNA were purchased from IDT (Integrated DNA Technologies, Coralville, IA) and were used without purification unless otherwise noted. See Supplementary Methods for a list of all other chemicals and their suppliers as well as for the synthesis of the GDGEGDGEGD peptide (Supplementary Fig. S8). The activated ribonucleotide monomers guanosine 5′-phosphor-2-methylimidazolide (2-MeImpG) and cytidine 5′-phosphor-2-methylimidazolide (2-MeImpC) were synthesized according to published procedues[58] with minor modifications (Supplementary Methods).

Experiments, Data Analysis, and Figures

All experiments were performed in triplicate or greater. Curve fitting was performed using MATLAB (Natick, MA) and figures were prepared with Igor Pro (Wavemetrics, Lake Oswego, OR) and Adobe Illustrator (San Jose, CA).

Fluorescence Anisotropy

A 2-aminopurine[37] (2AP)-containing RNA 15-mer (5′-CC(2AP)GUCAGUCUACGC-3′), 10-mer (5′-CC(2AP)GUCAGUC-3′), or 7-mer (5′-CC(2AP)GUCA-3′) was diluted to 10 μM in annealing buffer (10 mM Tris-Cl, 50 mM NaCl, and 1 mM EDTA in nuclease-free water at pH 8) to a total volume of 100 μL in a sample cuvette (Starna Cells, Inc., Atascadero, CA, 10 mm pathlength). We increased the concentration of peptide (R10NH2, R7, or R5) by adding small volumes (~1 μL) of concentrated solutions (100 μM–10 mM) to the sample cuvette and obtained data at each point (303 nm excitation, 370 nm emission) on a Cary Eclipse fluorescence spectrometer (Agilent Technologies, Santa Clara, CA). We modulated the magnesium and sodium concentration in the same way (Supplementary Fig. S4). See Supplementary Methods for further details.

Circular Dichroism

5 μM of an RNA 15-mer (5′-GCGUAGACUGACUGG-3′) or a 15-mer duplex (5′-GCGUAGACUGACUGG-3′ and its complement) in annealing buffer (10 mM Tris-Cl, 50 mM NaCl, and 1 mM EDTA in nuclease-free water at pH 8) was prepared in a 300 μL quartz cuvette (Starna Cells, 10 mm pathlength). Data was obtained using an Aviv 202 circular dichroism spectrometer (Aviv Biomedical, Inc., Lakewood, NJ) from 200 nm to 350 nm with a 2 nm step size and an 8 nm bandwidth. See Supplementary Methods for further details.

Stopped-Flow Annealing Experiments

A typical stopped-flow annealing experiment consisted of mixing two RNAs, e.g. one 15-mer (5′-GCGUAGACUGACUGG-3′) and one fully complementary 2AP-containing 15-mer (5′-CC(2AP)GUCAGUCUACGC-3′). All 2AP bases were deoxyribonucleotides, as provided by IDT. 1 μM of each RNA strand was diluted in annealing buffer (10 mM Tris-Cl, 50 mM NaCl, and 1 mM EDTA in nuclease-free water at pH 8) and incubated separately at room temperature for at least one hour either in the presence of or absence of peptide (R5, R7, R9, or R10NH2, up to 100 μM). The peptide length and concentration, as well as the length of one of the strands of RNA, was varied as indicated in Fig. 3. The two solutions were injected into the mixing chamber of an SX20 stopped-flow spectrometer (Applied Photophysics, Leatherhead, Surrey, UK), and the spectral data was immediately recorded (303 nm excitation, WG 320 nm emission filter). See Supplementary Methods for further details.

Nonenzymatic Primer Extension

Nonenzymatic RNA primer extension reactions were performed with 2-MeImpG. See Supplementary Methods for further details.

Confocal Fluorescence Microscopy

All samples were imaged using a Nikon (Tokyo, Japan) A1R confocal microscope (100×, 1.49 N. A. Apochromat TIRF oil immersion objective) microscope at 561 nm (pinhole 0.5 AU). See Supplementary Methods for further details.