Author response: Nonenzymatic copying of RNA templates containing all four letters is catalyzed by activated oligonucleotides

Field: Chemistry (Physical Sciences)

Full text Figures and data Side by side Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract The nonenzymatic replication of RNA is a potential transitional stage between the prebiotic chemistry of nucleotide synthesis and the canonical RNA world in which RNA enzymes (ribozymes) catalyze replication of the RNA genomes of primordial cells. However, the plausibility of nonenzymatic RNA replication is undercut by the lack of a protocell-compatible chemical system capable of copying RNA templates containing all four nucleotides. We show that short 5′-activated oligonucleotides act as catalysts that accelerate primer extension, and allow for the one-pot copying of mixed sequence RNA templates. The fidelity of the primer extension products resulting from the sequential addition of activated monomers, when catalyzed by activated oligomers, is sufficient to sustain a genome long enough to encode active ribozymes. Finally, by immobilizing the primer and template on a bead and adding individual monomers in sequence, we synthesize a significant part of an active hammerhead ribozyme, forging a link between nonenzymatic polymerization and the RNA world. https://doi.org/10.7554/eLife.17756.001 eLife digest Though defining what makes something “alive” has proved challenging, one crucial feature of living things is the ability to copy genetic information and pass it on to the next generation. Nowadays, enzymes called polymerases copy genetic information encoded within the DNA of living cells. However, when life on Earth began approximately four billion years ago, polymerases had not evolved yet. This means that the genetic information of the first cells had to be copied some other way. The earliest life on Earth is unlikely to have used DNA to store its genetic information, and probably used a closely related molecule called RNA instead. Like DNA, RNA is made up of four smaller building blocks joined together to form long chains. The building blocks of RNA are commonly referred to using single letters: A, C, G and U. Previous studies have shown that it is possible to copy RNA without enzymes, but for only two of the four RNA letters, namely C and G. Prywes et al. wanted to know if it was possible to create a chemical system, without polymerases, in which all four RNA letters could be copied. The experiments showed that strings of RNA that were three letters long could catalyze RNA copying, just as long as they were chemically activated. That is to say, these short RNA strings allowed RNA to be copied without enzymes if they had a chemical group at one end that made them more reactive.Each short catalyst helped copy one of the four RNA letters, and adding several into one reaction meant that longer sequences containing all four RNA letters could be copied. Prywes et al. then used these short catalysts to copy an RNA molecule that itself acts a bit like an enzyme, and confirmed that a significant portion of this molecule could be copied without any polymerases. Further work is now needed to see if it is possible to copy other RNA sequences, and especially longer ones, without enzymes. Another challenge for the future would be to attempt to copy an RNA sequence multiple times without enzymes; a challenge that the earliest ancestors of cells on Earth must have overcome to pass their genetic information down through the generations. https://doi.org/10.7554/eLife.17756.002 Introduction In order to serve as a bridge from prebiotic chemistry to the RNA world (Patel et al., 2015; Powner et al., 2009), the nonenzymatic copying of RNA templates by suitably activated nucleotides (Figure 1a) must occur quickly enough to replicate functional RNA sequences faster than they degrade. However, the nonenzymatic copying of sequences containing all four nucleotides has not yet been possible for a number of reasons (Szostak, 2012), most notably the slow rate of primer extension with adenosine and uridine monomers (Joyce et al., 1987; Wu and Orgel, 1992b; Deck et al., 2011). Indeed, two adjacent A or U residues in the template will stop polymerization entirely (Wu and Orgel, 1992b) under normal conditions; even partial copying requires extreme conditions, such as the eutectic phase of frozen samples (Vogel and Richert, 2007), which are not compatible with replication within protocells. Extensive optimization of reaction conditions with the aim of improving the rate of primer extension with all four monomers, including varying the choice of divalent cation and leaving group, improved the rate and regiospecificity of polymerization for G and C but not A and U (Wu and Orgel, 1992b, c; Deck et al., 2011; Inoue and Orgel, 1981; Hagenbuch et al., 2005; Lohrmann et al., 1980). Recent biophysical studies suggest that low A and U monomer affinity for the template is not the only reason for this problem (Szostak, 2012; Izgu et al., 2015) since A binds only three-fold more weakly than G, but primer extension with A is at least 100-fold slower (Joyce et al., 1987; Wu and Orgel, 1992b; Deck et al., 2011; Heuberger et al., 2015) than with G. Moreover, Deck et al. have shown that downstream helper oligonucleotides (Deck et al., 2011) that provide incoming monomers with an additional binding surface improve the binding of A and U monomers, but the underlying difference in rates remains. The use of helper oligonucleotides allowed primer extension to proceed on a template containing all four bases over the course of several weeks, where previously such templates could not be copied at all. Figure 1 with 2 supplements see all Download asset Open asset Catalysis of nonenzymatic primer extension by activated downstream nucleotides. (a) Structure of 2-methylimidazole-activated guanosine-5′-monophosphate, and its schematic representation. (b) Schematic of the RNA primer extension reaction. N1 represents an individual ribonucleotide monomer in position to react with the primer, N2 represents either a monomer or an oligomer downstream, with a leaving group capable of interacting with N1. Dashed lines: Potential interactions between leaving groups or between the downstream leaving group and the upstream nucleotide N1. (c) Schematics of the RNA primers, templates, monomers and oligomers used in d–f. Templates are complementary to the displayed monomers and oligomers. Template 2 has a U following the C to which the G monomer is bound to prevent downstream binding of G. (d) Primer extension by polymerization or ligation on templates 1 or 3, respectively. Fits describe ln(fraction primer remaining) vs time, giving an apparent first-order rate constant (Figure 1—figure supplement 1). (e) Primer extension assay for the experiments described in c, showing reaction progress after 10 min. In lane 6, a primer + 4 band can be observed, representing the slow addition of the activated trimer after the monomer. (f) Pseudo-first order rates of the reactions described in c. Error bars indicate S.E.M; all experiments were performed in triplicate or greater. Reaction conditions: 10 μM primer, 11 μM template, 200 mM CHES pH 9.0, 200 mM MgCl2, 50 mM monomer, 1 mM trimer. https://doi.org/10.7554/eLife.17756.003 Here we show that the presence of a leaving group at the 5′ end of a helper oligonucleotide can be a source of significant catalysis. Activated helper oligonucleotides facilitate the addition of all four RNA monomers individually and in sequence in a single reaction. Finally, when a template is immobilized by attachment to a bead, sequential addition of activated monomers and helper oligonucleotides successfully promoted the synthesis of a significant part of an active hammerhead ribozyme. Results Given the historical and continuing problems in demonstrating efficient nonenzymatic template directed primer extension, one might wonder whether this biologically inspired model is appropriate for replication within primordial cells. An alternative scenario that might seem more reasonable in the context of prebiotic chemistry involves template copying by the initial formation of short oligonucleotides, followed by ligation events that generate longer oligonucleotide intermediates and eventually a full length copy (Szostak, 2011; James and Ellington, 1999). To test the viability of this hierarchical assembly model, we compared the rate of primer extension with activated monomers to the rate of ligation of two template bound oligonucleotides (Figure 1b,c reactions 1 and 3). Nucleotides activated with 2-methylimidzole on the 5′-phosphate have been used extensively to model nonenzymatic template copying (Deck et al., 2011; Wu and Orgel, 1992c), irrespective of their prebiotic plausibility, and were used in all experiments reported here. In both the nucleotide polymerization and the oligonucleotide ligation reactions, the chemical reaction is the same, i.e. attack of the 3′-hydroxyl of an oligonucleotide on the 5′-phosphate of a downstream nucleotide, with displacement of the 2-methylimidazole leaving group (Figure 1b). Moreover, the reacting nucleotides are the same in both cases: a G at the 3′-end of the primer and a G monomer or a G at the 5′-end of an oligonucleotide. We expected the ligation reaction to be faster, because the oligonucleotides to be ligated are both stably bound to the template, and because the fully base-paired nicked duplex should be largely in the optimal A-type conformation (Kozlov and Orgel, 2000), whereas the primer/template complex with template bound monomers would be significantly more disordered. To our surprise, the primer extension reaction proceeded almost 100 times more rapidly than the ligation reaction (Figure 1d). This was true for a variety of trimer sequences (Figure 1—figure supplement 2). In our search for an explanation for this unexpected observation, we reasoned that one of the major differences between the two scenarios was the presence of multiple adjacent 5′-activated nucleotides downstream of the primer in the case of monomer addition, but not ligation, where there is only a single activated nucleotide. Indeed, the case for a role of a second downstream activated nucleotide is consistent with the well known difficulty of extending a primer to the last nucleotide of a template (Wu and Orgel, 1992c). Furthermore, Orgel et al. showed over 20 years ago that efficient primer extension requires the presence of two activated monomers adjacent to the primer. Remarkably, the reaction is fastest when both monomers are activated with 2-methylimidazole instead of either or both being activated with imidazole (Wu and Orgel, 1992a), suggesting a possible catalytic role for a physical interaction of the leaving groups of adjacent monomers. In order to directly test the possibility that a 2-methylimidazole leaving group on the 5′-phosphate of a downstream monomer or oligonucleotide (Figure 1b) would increase the rate of reaction between a primer and an adjacent monomer, we designed a series of templates for nonenzymatic RNA polymerization and ligation (Figure 1c). We then measured the rate of addition of a monomer to a primer in the presence or absence of downstream nucleotides, with or without 5′-activation of the downstream nucleotides (Figure 1c–f). Rates were calculated assuming a pseudo-first order rate equation (Figure 1—figure supplement 1), because the concentrations of primer and template were far below that of the monomer, which would not change significantly during the reaction. Primer extension by addition of a single monomer, in the absence of any additional downstream mono- or oligo-nucleotides, was very slow and comparable in rate to the ligation of the primer to a 5′-activated GAG trinucleotide (Figure 1c–f, reactions 2 and 3). The binding of an unactivated AGC trimer, with either a 5′ hydroxyl or a 5′ phosphate, downstream of an activated G monomer conferred only a modest increase in the rate of addition of the G monomer to the primer (Figure 1c–f, reactions 4 and 5), consistent with previous reports of unactivated downstream ‘helper’ oligonucleotides (Deck et al., 2011; Jauker et al., 2015; Kervio et al., 2010). Such helper oligonucleotides are thought to act largely by increasing monomer affinity to the primer/template complex by providing an additional base-stacking surface. In contrast, when the same AGC trimer was activated as a 5′-phosphoro-2-methylimidazolide and used as a downstream helper oligonucleotide, the rate of primer extension (by addition of a G monomer) was increased by over two orders of magnitude (Figure 1c–f, reaction 6) compared to reactions with unactivated trimers. The rate of primer extension with a single G in the presence of an activated downstream trinucleotide was about twice as fast as the rate when the primer was followed by up to four sequential activated G monomers (Figure 1c–f, reaction 1 and 6). To systematically study the rate of nonenzymatic primer extension as a function of the length of the downstream activated ‘helper’ oligonucleotide, we synthesized a series of activated oligonucleotides of different lengths (Figure 2a). At saturating concentrations (Figure 2—figure supplement 1) of each of the oligonucleotides, the rate of primer extension improved as the length of the helper increased from mononucleotide to dinucleotide to trinucleotide, and then stayed approximately constant with the tetranucleotide (Figure 2a). At subsaturating concentrations of helper oligonucleotide, the rate of the primer extension reaction increased with increasing concentrations of di- or tri-nucleotide helper (Figure 2—figure supplement 1). Because the maximum rate of the trimer-assisted reaction was significantly faster than that of the dimer-assisted reaction, we proceeded with trimers as the downstream helper oligonucleotides for the remainder of this study. Figure 2 with 1 supplement see all Download asset Open asset Nonenzymatic primer extension using all four monomers. (a) RNA primer extension assay using alternative 'helper' oligomers and corresponding, complementary templates. The respective rates are shown at bottom. Concentration curves are provided for AG and AGC in Figure 2—figure supplement 1. (b) Primer extension assay for each of the four monomers, as well as 2-thiouridine, in the absence and presence of activated trimer. Shown at bottom of both a and b is a bar graph with the respective rates. Reaction conditions: 10 μM primer, 11 μM template, 200 mM Tris pH 8.0, 100 mM MgCl2, 50 mM monomer, 10 mM 2-MeImpAG, 1 mM 2-MeImpAGC, 1 mM 2-MeImpAGGC. The gels in a and b show reaction progress after 10 min and 1 hr respectively. Error bars indicate S.E.M; all experiments were performed in triplicate or greater. https://doi.org/10.7554/eLife.17756.006 We then tested the ability of a downstream 2-methylimidazole-activated AGC trimer to catalyze template-directed RNA primer extension with all four individual 2-methylimidazole activated monomers – A, G, C and U. We also tested the 2-thiouridine monomer, which we have previously shown to be superior to uridine in nonenzymatic primer extension in terms of both increased rate and improved fidelity (Heuberger et al., 2015). In each case, the rate of primer extension increased by at least two orders of magnitude compared to the corresponding reactions without any helper oligonucleotides (Figure 2b). Remarkably, whereas the rate of primer extension with A and U monomers was previously too slow to measure (Wu and Orgel, 1992b; Heuberger et al., 2015) – conservatively estimated to be at least three orders of magnitude slower than primer extension with G or C monomers – the difference in polymerization rates was reduced to roughly one order of magnitude when assisted by downstream activated trimers (Figure 2b). In order to assess the fidelity of nonenzymatic trimer-assisted primer extension, we measured the rates of monomer addition for all monomer-template combinations, including both matches and mismatches (Figure 3a). We calculated the fidelity for each template as the rate of matched monomer addition divided by the sum of matched and mismatched monomer addition rates. By averaging the fidelities of the four templates we found the overall fidelity of trimer-assisted polymerization to be 98%. Assuming an error threshold of one mutation per genome (Eigen, 1971), this value of fidelity allows for effective genome sizes of 50 nucleotides, long enough to produce functional ribozymes (Ferré-D'Amaré and Scott, 2010). The fastest mismatch reaction was primer extension by G during copying of a template U, which was approximately 5% as fast as primer extension with A on the same template (Figure 3a, Figure 3—figure supplement 1). This is likely due to the formation of a G:U wobble base pair (Heuberger et al., 2015), and is consistent with previous studies of nonenzymatic primer extension reactions (Rajamani et al., 2010). All other mismatches were at least two orders of magnitude slower than their matched counterparts. We also tested the effect of replacing the canonical U monomer with 2-thiouridine and 2-thioribothymine on the polymerization rate and found, in line with previous reports (Heuberger et al., 2015), that this sulfur substitution improved the rates by approximately one order of magnitude (Figure 3b), bringing the rate of primer extension closer to the rates of G and C addition. More rigorous tests of fidelity, in which monomers can compete for binding on a template in all possible sequence contexts, are ongoing. Figure 3 with 1 supplement see all Download asset Open asset Fidelity of trimer-assisted primer extension. (a) Schematic of the fidelity assay. Using the same RNA primer and trimer, four different templates, one with each base, were paired with each of the four 2-methylimidazole activated monomers to test the relative rates of each matched and mismatched pair. (b) Heat map showing the relative rates of primer extension for each template:monomer pairing. Black indicates a rate of 0.001 h−1, white indicates a rate of 100 h−1. A bar graph and a table of the same data can be found in Figure 3—figure supplement 1. (c) Relative rates of primer extension with the modified U monomers, 2-thiouridine and 2-thioribothymidine compared to that of a canonical, activated U monomer on a template containing an A. Reaction conditions as in Figure 2. Error bars indicate S.E.M. All experiments were performed in triplicate or greater. https://doi.org/10.7554/eLife.17756.008 Encouraged by the relatively fast and accurate addition of all four monomers to a primer, we then attempted to copy short RNA templates containing all four nucleotides. In order to iterate the process of trimer assisted monomer addition, after a downstream activated trimer catalyzes the addition of the first monomer to the primer, the helper trimer must dissociate to allow for the binding of the next monomer-trimer pair. To test the feasibility of this mode of primer extension, we designed a template that contained binding sites for all four monomers and synthesized the appropriate activated trimers (Figure 4a). We used 2-thiouridine monomer in place of uridine (see above), and all four monomers (A, G, C and 2-thiouridine) were present in every reaction. In the absence of activated trimers, we observed almost no primer extension (lane 1 Figure 4b); even though all four monomers were present, their combined rate of addition was not measurable. Adding just the first helper trimer resulted in rapid addition of the first monomer (C) to the primer, followed by slow addition of the first trimer (AGC) to the growing primer. Similarly, adding two, three or four trimers together resulted in the generation of primers extended by two, three or four monomers respectively (Figure 4b). These reactions converted primer into a product with four sequentially added monomers with an ~80% yield after 16 hr (Figure 4c,d); single monomer addition reactions reached >95% yield after only 10 min (Figure 2b). The difference between the fast rates observed in the trimer-assisted addition of a single base (Figure 2b) and the slower rate of trimer-assisted polymerization of all four bases simultaneously (Figure 4b) may be due to the competition of trimers for overlapping binding sites on the template. This hypothesis is supported by the decreased rate of trimer assisted primer extension with a single monomer in the presence of additional overlapping downstream trimers (Figure 4—figure supplement 1). The copying of templates containing two consecutive A or U monomers is also made possible by the use of activated helper oligonucleotides (Figure 5). Figure 4 with 1 supplement see all Download asset Open asset Primer extension with all four monomers in a one-pot reaction. (a) Schematic of a primer extension reaction incorporating all four RNA monomers, with four respective downstream helper trimers. (b) All four monomers and the activated trimers indicated below the gel were added to each reaction. For example, in lane 3 all four monomers and the activated AGC and GCG trimers were mixed with primer, template, buffer and Mg2+. Higher bands represent either the ligation of activated trimers or the polymerization of G monomers without trimer assistance. The gel shows reaction progress after 16 hr. (c) Timecourse of the reaction with all four trimers. A 48 hr timepoint of the reaction without any trimers (left lane of b) is contrasted with a timecourse (timepoints at 5 and 30 min, 2, 24 and 48 hr). The percentage of polymerization products that have been extended by at least four nucleotides is plotted over time. (d) A gel the plotted in part c. A timecourse showing the effect of overlapping trimers on reaction rate can be found in Figure 4—figure supplement 1. Reaction conditions: μM primer, μM template, 100 mM pH 8.0, 100 mM MgCl2, 20 mM monomer, 100 μM trimer. Figure 5 Download asset Open asset of primers by multiple consecutive A or U nucleotides. (a) Schematic of a primer extension reaction two consecutive A nucleotides can The gel shows extension without trimers and with one or two trimers after 16 hr. (b) as in (a) but with two U In the is with nonenzymatic RNA polymerization the RNA then it would have been to synthesize RNA sequences long enough to function as ribozymes (Deck et al., 2011). our can only produce sequences nucleotides in one reaction in low (Figure supplement 1) due to the effect of trimers with overlapping binding sites (Figure 4—figure supplement 1), we reasoned that a could be synthesized by this if monomers were with their trimers, one at a (Figure 6). To that we immobilized a template that for the sequence of one of the hammerhead – on a bead and added matched of monomers and trimers sequentially (Figure Primer extension was by gel the that no 2-thiouridine monomers were used order to the catalytic of the a rate was observed at each (Figure The reaction to these rates for example, the monomer addition hr to the addition reached the same of after just 30 min. G and C were faster than A or U the reason for the between the rates of these reactions on and in is The use of or might some of the The product was from the bead by of an of a DNA oligonucleotide of sequence to the immobilized template. the hammerhead was added to the synthesized of oligonucleotides in the presence of 200 mM for 16 hr (Figure the that this was a reaction with only of the reaction the product was to function as a with demonstrating that a significant portion of an active was synthesized Figure with 1 supplement see all Download asset Open asset Nonenzymatic RNA synthesis of an active hammerhead by template primer extension. (a) Schematic of the primer extension reaction. of the hammerhead by the sequential addition of matched of activated monomers and trimers. (b) lane shows primer extension by one nucleotide, catalyzed by the corresponding downstream activated trimer. yield of the product was Reaction conditions: 100 mM MgCl2, 200 mM pH mM monomer and 2 mM trimer. The were between in 2 1 mM and 10 mM pH For 2, 5 and 50 mM monomer and 4 mM trimer were was by gel it reached in length from 30 min to the was hr. (c) The product of the last reaction was from the bead by the addition of the an of the DNA sequence of the extended primer at over 2 min. the hammerhead was The lane shows the of the reaction, after 16 hr at For made from DNA and RNA as and respectively. Reaction conditions: primer from the was added to 200 mM MgCl2, 200 mM pH and Discussion The experiments described for the first a and means of copying mixed sequence RNA templates. activated oligonucleotides – by either or monomer polymerization – are efficient catalysts of fidelity primer extension with all four RNA monomers. short activated oligonucleotides can either as catalysts of by monomer addition, or directly as replication since they can also be into a growing by Template copying in a complex but containing both activated monomers and oligomers could occur a process primer extension with monomers and oligonucleotide The in rates between the reactions catalyzed by activated unactivated trimers indicates a role for the leaving group downstream of the monomer. The slow rate of activated oligonucleotide ligation is also by the lack of a leaving group one base downstream of the of the primer extension in this case the downstream position to the second monomer in the oligonucleotide which has no leaving group as it is to the upstream monomer by a The of the interaction between the downstream leaving group and the reacting monomer is possible that the downstream leaving group with the reacting leaving group, it for attack by the hydroxyl of the primer. observed by Wu et al. (Wu and Orgel, this interaction could be very to such that 2-methylimidazole at the downstream position would facilitate polymerization more than though the that such are not likely to the of the Wu et al. (Wu and Orgel, also that the catalytic effect could be due to catalysis. Another possibility is that the upstream leaving group a with the of the downstream monomer, a reaction that is only possible if there is a leaving group on the downstream monomer et al., and and This could form the attack of the hydroxyl of the primer, an or in a reaction where the leaving group of the upstream monomer the leaving group of the downstream monomer. Further of the reaction could to in the rate and of template Further are to the copying of longer and functional RNA sequences in one protocell-compatible However, the copying of long mixed template sequences under our conditions both in terms of rate and with reactions in which RNA templates are copied by evolved polymerases et al., 2011) (Figure supplement 1). In if the rates and fidelities of nonenzymatic RNA polymerization were the for a in the RNA world may be RNA could be copied by