The phenotypic richness of a biopolymer pool, that is the density of functional sequences within the sequence space (the totality of all available or possible sequences), is a central, if largely unexplored parameter of its evolutionary potential. This property critically informs many aspects of biological function including the adaptability of biological systems as a function of combinatorial diversity ranging from bacterial populations to the mammalian immune system and to abiotically-generated sequence pools. In all such cases, ‘survival’ (in a Darwinian sense) depends on the availability of functional entities (such as a neutralizing antibody) within the combinatorial space available to the system (such as the number of circulating B-lymphocytes or the size of a phage display repertoire). The same concept is also of central importance to the proposed spontaneous emergence of function from prebiotic pools of informational polymers, a key conjecture of current scenarios for the origin of life. Indeed, the phenotypic richness of random-sequence pools of the natural nucleic acids RNA and DNA, as well as a variety of xeno nucleic acids (XNAs), has been studied by in vitro selection methods reliant on iterative rounds of enrichment and amplification, yielding ligands (aptamers) and catalysts (Wachowius et al., 2017). Extrapolation from such selection experiments and analysis of the mutational landscapes of functional oligomers indicated a low frequency of such functional molecules (1 in 10¹⁰-10¹³) (Wilson and Szostak, 1999), suggesting that, globally, the pools are essentially inert, although weak ligands/catalysts might be more frequent (Ekland et al., 1995; Knight et al., 2005). Together, experimental data and theoretical considerations suggest that function scales with sequence (i.e. information) and structural complexity (or a related property, termed ‘functional information’ (Carothers et al., 2004)). Thus, sequences with any given function (and among these, those with highest functionality) are more frequently found in longer, informationally and compositionally more complex, and by inference structurally more diverse pools, despite a potential trade-off through increased misfolding (Ekland et al., 1995; Sabeti et al., 1997; Carothers et al., 2004; Legiewicz et al., 2005; Jiménez et al., 2013). In agreement with this, in silico models predict a sharp drop in the propensity of both folded structures and structural complexity in RNA pools with shorter oligomer lengths (<25 nucleotides, nt) (Briones et al., 2009). Consequently, the pools of short, random RNA oligomers of mixed sequence composition (<20 nt) accessible from prebiotic chemistry (Monnard et al., 2003) would be predicted to contain few defined three-dimensional structures and even fewer functional sequences. This raises the question how functions that would constitute a living system could ever have emerged spontaneously from such pools.

Starting from the premise that the global potential of oligomer pools for function may have been underestimated by an undue focus on single ‘hit’ sequences (i.e. fitness peaks), we have sought to examine the innate, global functional potential of the pools themselves, which has never been measured directly. Specifically, we examine pools of short RNA oligomers of semi- and fully-random sequence (with or without activation chemistry) and compare them to equivalent pools of chemically related but distinct genetic polymers, including DNA as well as the xeno nucleic acids (XNAs) ANA (arabino-), HNA (hexitol-) and AtNA (altritol-nucleic acids), with regards to a simple functional test: the ability to undergo intermolecular ligation and/or recombination. While DNA, ANA and HNA pools proved inert, we discover a pervasive tendency of diverse-sequence RNA (and to a lesser extent AtNA) pools to undergo ligation and/or recombination even in the absence of extraneous activation chemistry, and analyse the constitution and emergent properties of recombined pools by deep sequencing, bioinformatic analysis and in silico simulation. Our results suggest that RNA (and to a lesser extent AtNA) hold a privileged position among simple alternatives for genetic information storage and propagation due to an innate capacity for recombination enabled by energetically near-neutral transesterification chemistry (Eftink and Biltonen, 1983; Usher and McHale, 1976; Verlander et al., 1973; Verlander and Orgel, 1974). Furthermore, our results demonstrate that such spontaneous, non-enzymatic recombination among random-sequence pool members acts to counterbalance hydrolytic decomposition and in the process, progressively increases pool compositional, informational and structural diversity.

We chose to specifically examine 10¹⁵-member pools of eicosamer (20 nt long) RNAs. While still within the range of oligomer lengths likely to be accessible via prebiotic chemistry (Ferris et al., 1996; Monnard et al., 2003), 20-mer sequences are predicted to exhibit at least some tendency for secondary structure formation (Briones et al., 2009). Furthermore, a 10¹⁵-member eicosamer pool (1.7 nmol) comprises significant redundancy (909 copies) of each possible 20-mer sequence. Finally, as shown previously, naturally occurring 20-mer sequences can associate into non-covalent multimer complexes reconstituting function such as catalysis, when incubated in eutectic ice phases at −9 °C, as exemplified by the hairpin ribozyme (Mutschler et al., 2015; Vlassov et al., 2004).

In order to examine the innate potential of such pools for functionality (such asligation), we first examined the reactivity of 2’, 3’-cyclic phosphate (>p)-activated random RNA pools. >p groups form as part of RNA degradation reactions, as well as during proposed prebiotic nucleotide synthesis (Jarvinen et al., 1991; Li and Breaker, 1999; Powner et al., 2009; Gibard et al., 2018) and can mediate non-enzymatic ligation in the presence of an organizing template (Lutay et al., 2006; Usher and McHale, 1976). In order to maximize reactivity, we incubated pools in ice; eutectic ice phase formation has been shown to promote molecular concentration, reduce RNA hydrolysis, and provide a benign, compartmentalized medium, making water ice a potentially beneficial medium for RNA propagation at the origin of life (Attwater et al., 2010; Attwater et al., 2013; Mutschler et al., 2015). We first incubated a >p activated random RNA eicosamer pool (N₂₀>p, including 6% 5’-Carboxyfluorescein (FAM) labelled N₂₀>p to facilitate detection of ligation products) in ice without any organizing template and sought to detect higher molecular weight (MW) product bands by gel shift in denaturing gel electrophoresis (Urea-PAGE) (Figure 1A). We observed significant intermolecular, covalent N₂₀>p pool ligation with yields of ~10% (after 2 months, −9 °C) both in the presence or absence of magnesium (+ / - Mg²⁺) counterions (10 mM MgCl₂, Supplementary file 1). No ligation was observed in equivalent control samples incubated at −80 °C (Figure 1B). Deep sequencing of the most prominent higher MW band confirmed that >80% of sequences corresponded to ligation events between two eicosamers (N₂₀ × N₂₀, Figure 1C). Thus, RNA eicosamer pools activated by >p undergo a disproportionation reaction by intermolecular ligation, yielding a subpopulation of pool sequences extended in length (predominantly 40-mers and some 60-mers (Figure 1C)).

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Comparison of the 40mer ligation product sequences with the sequence distribution of pre-ligation eicosamers revealed a clear nucleotide signature at the N_-1pN₊₁ ligation junction (predominantly CpN, with UpG also overrepresented) (Figure 1D, Figure 1—figure supplement 1A). While the mechanistic basis for this bias remains to be elucidated, we hypothesize that the stable pairing and the slightly higher reactivity of pyrimidine nucleoside 3′-phosphodiesters (hydroxide ion catalysed cleavage of 3′, 5′-YpN is ca. 2x-3x faster than 3′, 5′-RpN cleavage (Oivanen et al., 1998)) aides >p dependent ligation. Regardless of the mechanism, the resulting sequence fingerprint allows an assignment of ligation junctions and reaction trajectories proceeding through an analogous, putative >p like intermediate or transition state in diverse RNA contexts (see below).

Other global features of the ligation junction are notable. In silico RNA folding using RNAfold (Lorenz et al., 2011) comparing experimental versus computer-generated sequence pools using the same post-ligation nucleotide distribution (shown in Figure 1D) indicates a significant enrichment of secondary structure in the ligated pool with ca. 55% of intermolecular ligation junctions located within helical regions (Figure 1E). In accordance with previous studies on >p reactivity, this suggests that ligation is accelerated by inter-strand hybridization, which positions reactive groups in proximity and increases their effective concentration, while reducing the entropic cost of intermolecular ligation. In support of this, we found evidence for extensive intramolecular association and non-covalent complex formation in random eicosamer pools as judged by non-denaturing gel electrophoresis (Figure 1—figure supplement 2). Thus, random pools contain a significant proportion of organizing templates through intermolecular hybridization among pool oligomers and this provides gapped duplex junctions poised for ligation (Lutay et al., 2006; Usher and McHale, 1976).

We wondered if these pools contained RNA sequence motifs that would promote ligation by mechanisms other than gapped duplex formation. To aiddiscovery of specific motifs, we repeated the ligation experiments using semi-random RNA sequence pools comprising random eicosamers (N₂₀) flanked by (unstructured) 20 nt constant sequence primer binding sites (C1₂₀, C2₂₀). The use of such ‘bait’ (C1₂₀-N₂₀>p, B>p) and ‘prey’ (5’OH-N₂₀-C2₂₀, OH-P) oligonucleotides (2.5 µM each) enabled rapid detection and analysis of intermolecular bait × prey (B × P) ligation products with high sensitivity by RT-PCR using primers complementary to C1₂₀ and C2₂₀ as well as much shorter incubation times than needed for direct detection (Figure 2A,B and Figure 2—figure supplement 1A). Deep sequencing of RT-PCR products revealed ligation of semi-random RNA sequence pools (40 nt) into 80 nt B-P ligation products (5’-C1₂₀-N₂₀-N₂₀-C2₂₀-3’), with very similar features as in the fully-random pool, including the above described CpN ligation junction ‘fingerprint’ and the increased base-pairing bias at the ligation site (Figure 1—figure supplement 1B, Figure 2—figure supplement 1B). Indeed, when we randomly sampled clones from the ligated fraction and analysed the ability of the constituent B, P segments of the most active clone for ligation, activity depended on formation of a gapped duplex junction with distal sequences having little impact on ligation (Figure 2—figure supplement 2).

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Applying a more stratified analysis, we searched for other RNA motifs than a gapped duplex that may mediate RNA ligation by comparing ligated B-P-sequences from two different semi-random RNA pools (differing in their C₂₀ sequences, Supplementary file 2). The use of different C₂₀ anchor sequences (C3₂₀, C4₂₀) allowed us to dissociate motif structure from sequence-dependent effects such as the imprint of pairing with the conserved C₂₀ termini. We clustered ligation junction structures according to (predicted) minimal free energy using 408 different secondary structure sub-motifs based on 25 main-motifs (Motif A - Motif Y) including internal loops and hairpins (Figure 2—figure supplement 3). While ligation frequencies of most of these motifs broadly correlated with predicted base pairing frequency at the ligation junction (consistent with formation of a helical junction and proximity ligation, see above), we identified high ligation frequencies at specific positions in the unpaired regions of some predicted internal loop motifs (motif H and J, Figure 2C,D). The consensus sequence of these submotifs, H4 and J4, could subsequently be extracted using the motif discovery tool DREME (Bailey, 2011). In both motifs, internal loop segments are formed opposite the putative ligation junctions with involvement of sequences from the C₂₀ constant regions, which seemed to enhance ligation (and therefore enrichment) of compatible junctions from a subpopulation of random segments (Figure 2E,F).

Strikingly, motif J4 and H4 were sufficient to promote regioselective ligation in minimal hairpin constructs (Figure 2G,H, Figure 2—figure supplements 4 and 5): A minimized version of the J4 motif promoted reversible ligation via 2’−5’ regiochemistry both in ice (in the absence of Mg²⁺) and at ambient conditions (strictly Mg²⁺-dependent) with an up to 10-fold enhanced apparent ligation rate (k_obs) compared to a simple gapped duplex reaction under the same conditions (Figure 2—figure supplement 6). A search for the J4 consensus motif in the JAR3D structure database (Zirbel et al., 2015) revealed that J4-like motifs are present in in naturally occurring RNAs such as the domain II of the 23S rRNA of Deinococcus radiodurans (Harms et al., 2001) and the Escherichia coli adenosylcobalamin riboswitch (Johnson et al., 2012), for which structural information is available. In both structures, the J4-like motifs form a stable pseudohelical structure comprising a purine-rich 4 × 4 internal loop with a triple-sheared GA motif that immediately suggests a likely catalytic involvement of a cross-strand adenine in the reversible 2’−5’ ligation reaction (Figure 3). By contrast, no structural information is currently available for the H4 motif, which mediates ligation via canonical 3’−5’ regiochemistry (exclusively under eutectic ice conditions (−9 °C) in the absence of Mg²⁺) albeit with a 2- to 3-fold slower k_obs than an equivalent gapped duplex-mediated reaction (Figure 2—figure supplement 4, Figure 2—figure supplement 6). Thus, random pools comprise a wide variety of RNA motifs capable of promoting >p ligation via either 2’−5’ or 3’−5’ regiochemistry.

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Next, we sought to dissect mechanistic modes of intermolecular RNA ligation using binary combinations of semi-random RNA bait x prey (B × P) sequence pools (2.5 µM each) each bearing different 2’, 3’- or 5’-substituents (Figure 4A). As described above, we observed efficient and rapid ligation of B>p × 5’OH-P pools (and no ligation in negative controls incubated at −80 °C), as judged by RT-PCR (Figure 4B). Furthermore, we also observed intermolecular assembly in 2’- (or 3’-) phosphorylated B-2’p (or B-3’p) × 5’OH-P reactions. However, these predominantly yielded shorter products, varying between 40–80 nt in length (Figure 4B,C), while still displaying the characteristic CpN ligation ‘fingerprint’ suggestive of reaction via a >p intermediate (Figure 4D, Figure 1—figure supplement 1E). Thus, the CpN sequence signature allowed mapping of presumed ligation junctions, which revealed variable-length assembly products derived from progressive truncation of bait segments, while preserving prey segment length (Figure 4D, Video 1), suggesting a mechanism involving intermolecular recombination (rather than ligation). Recombination likely proceeds via a transesterification mechanism involving a nucleophilic attack of the prey 5’-OH on bait segments either directly (leading to internal bait segment cleavage) or on the >p of bait segments truncated by hydrolysis (Figure 4—figure supplement 1). Consistent with this mechanistic hypothesis, blocking the prey pool 5’-OH by phosphorylation (5’p-P, Figure 4A,B) abolished recombination (Figure 4B,C). Furthermore, qRT-PCR suggests that recombination proceeds with a >10 fold slower rate than direct >p dependent ligation (Figure 4—figure supplement 2), consistent with a putative multi-step reaction trajectory involving a slow, hydrolytic generation of truncated, activated bait (B>p) intermediates for ligation, as well as potentially direct in-line attack in a subset of reactions (Figure 4—figure supplement 1).

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In order to explore the consequence of alternative activating groups, prey pool activation with a 5’-triphosphate (5’ppp-P) was also examined (Figure 4—figure supplement 3). These yielded an even broader length distribution of products, comprising full-length 80 nt B-P ligation products (likely originating by direct nucleophilic attack of the bait pool terminal bait-3’OH (or −2’OH) attack on the 5’ppp of the prey pool), as well as a wide range of shorter products, derived from both B and P truncationlikely as a result of non-canonical, multi-step reaction trajectories. Indeed, we note that our method of RT-PCR amplification omits the detection of any potential intermolecular recombination or ligation product that does not display a B-P sequence arrangement. Therefore, bait or prey-pool self-ligation (or recombination) (yielding B-B, P-P type products) or reverse ligation (yielding P-B) or indeed non-canonical intramolecular reactions yielding circular, lariat or branched products (unless resolved into a B-P arrangement) are not detected (Figure 4—figure supplement 4). Thus, although rapid and convenient, our PCR assay likely underestimates the true level of intra- and inter-pool reactivity (and the true molecular diversity of reaction products).

Nevertheless, most significantly (and consistent with the reactivities observed above), we observed comparable intermolecular reactivity in unmodified semi-random RNA sequence bait and prey pools (C1₂₀-N₂₀-3’OH × 5’OH-N₂₀-C2₂₀) - that is pure RNA pools devoid of phosphorylation or any other activation chemistry. Remarkably, these showed intermolecular assembly, yielding a similar distribution of truncated products as observed for bait-2’/3’p×5’OH prey reactions, suggesting intermolecular recombination via transesterification chemistry as outlined above (Figure 4B,C).

In order to better understand the reactivity of unmodified RNA pools without the obscuring imprint of conserved C₂₀ sequences (and PCR amplification bias), we next sought to directly observe reactivity of random RNA eicosamer pools devoid of any activation chemistry. In these (bait × prey: 5’FAM-N₂₀-3’OH × 5’OH-N₂₀A₁₀-3’OH), the prey pool included an invariant A₁₀ tail sequence to aid recovery and analysis (by poly-dT primed reverse transcription), and to allow detection of prey self-recombination products (P-P). After an extended incubation (5 months, −9 °C) we were able to directly visualize formation of two distinct recombination products by gel electrophoresis without any amplification (Figure 5A). The two main product bands (Rec1 and Rec2, together ca. 2.3% yield) were analysed using either poly-dT-primed (for Rec1) or poly-U tailing followed by poly-dA primed SMARTer RT-PCR (Rec1 and Rec2) and deep sequencing (Figure 5—figure supplement 1). For Rec1, we found recombination fragment distributions from ~40 to ~50 nt that include the characteristic fingerprint of a >p ligation junction (Figure 5B,C). In contrast, the longer (on average) Rec2 products (~45 to ~55 nt) showed an ApN ligation junction as they were formed (albeit less efficiently (ca. 0.2% yield)) from recombination of two prey segments within the A₁₀ tail (Figure 5B,C). Thus, as with the unmodified semi-random sequence RNA pools (Figure 4B,C), intermolecular ligation products in random, non-activated RNA pools are likely formed by a transesterification process initiated either by direct recombination or by hydrolytic cleavage (of either B or P), followed by putative ligation of truncated RNA>p sub-fragments. These mechanisms give rise to recombination product species such as bait-prey (B-P, forming Rec1) or prey-prey (P-P present in both Rec1 and Rec2). Bait-bait recombination (B-B) products are not observed, as the bait 5’OH is blocked by FAM modification to aid analysis.

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Comparison of Rec1 sequence pools with three random pools (3 × 300,000 sequences) of equal nucleotide distribution generated in silico again indicated that recombination sites were preferentially localized in base-paired (gapped duplex motif) regions (Figure 5D). Furthermore, selected pools not only exhibited an overall increase in local duplex formation, but also generally more stable (predicted) minimal free energy structures (Figure 5E). Thus, pure random-sequence RNA pools, devoid of any activation chemistry or conserved sequence regions (apart from an A₁₀ tag) display an innate capacity for intermolecular recombination, yielding subsets with altered oligomer length distribution (including longer RNA oligomers), enhanced sequence diversity and structural stability.

Finally, we sought to dissect critical chemical parameters required for an innate recombination potential. To this end, we expanded our investigation beyond RNA to compare and contrast the potential of pools of a range of RNA congeners (representing systematic variations of the canonical ribofuranose ring structure), with respect to intermolecular ligation or recombination. We first examined unmodified, semi-random sequence pools of DNA or prebiotically plausible ANA (arabino nucleic acid) (Noronha et al., 2000; Roberts et al., 2018). In these chemistries, the canonical ribofuranose ring of RNA is replaced with either a 2’-deoxyribose (DNA) lacking the 2’ hydroxyl of RNA, or an arabinofuranose ring (ANA), in which the analogous 2’-hydroxyl group is in the axial (trans) position. Like RNA, both DNA and ANA are capable of forming functional catalytic motifs (Silverman, 2016; Taylor et al., 2015), including small self-cleaving DNA catalysts (Gu et al., 2013). Therefore, both DNA and ANA pools contain sequences with potential function, and in the case of DNA should be able to perform autocatalytic DNA strand cleavage, as a first step of a recombination reaction. However, while we readily detected recombination in the semi-random-sequence RNA pools (as above), neither DNA nor ANA pools showed detectable recombination even after prolonged incubation (up to 6 months, −9 °C; Figure 6A) and despite the 10–100-fold higher sensitivity of PCR (DNA) compared to RT-PCR (RNA and ANA) detection (Ohuchi et al., 1998; Okello et al., 2010). Even inclusion of transition metal ions (Zn²⁺), which had been shown to strongly promote DNA self-cleavage (Gu et al., 2013), did not result in recombination in DNA (or ANA) pools, while apparently inhibiting it in RNA pools (Figure 6—figure supplement 1).

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Next, we explored the reactivity of pools of two additional RNA congeners, in which the five-membered ribofuranose ring is replaced by six-membered ring analogues: D-altritol nucleic acid (AtNA), which shares an analogous vicinal 2’, 3’-cis-diol configuration with RNA (Figure 6B), and its 2’-deoxy-analogue, hexitol nucleic acid (HNA). While HNA random-sequence pools had been shown to contain catalytic motifs (Taylor et al., 2015), AtNA random-pools had not previously been explored.

To test whether the AtNA vicinal hydroxyl can participate in transesterification reactions despite its different ring geometry, we first exposed defined-sequence HNA and AtNA (as well as equivalent RNA) oligonucleotides to alkaline pH (NaOH). Indeed, while HNA proved inert, AtNA (like RNA) proved to be sensitive to alkaline pH-induced hydrolysis, albeit with an apparent 6-fold slower strand scission rate than RNA (Figure 6—figure supplement 2). This indicates that (akin to RNA) the equivalent vicinal 2’-OH in AtNA can participate in intramolecular transesterification reactions presumably via an analogous cyclic phosphate (AtNA>p) intermediate (Figure 6—figure supplement 3). At the same time, the observed lower hydrolytic reactivity suggests that formation of AtNA>p cyclic phosphate may be energetically less favourable than in RNA. Next, we examined whether HNA and AtNA pools (devoid of extraneous activation chemistry) could participate in intermolecular recombination reactions. To this end, we compared the reactivity of semi-random sequence pools of HNA and AtNA to equivalent RNA pools, but observed unambiguous recombination only in the RNA pools (as seen previously) (Figure 6—figure supplement 4). To expedite potentially weak tendencies for recombination, we exposed semi-random sequence pools of HNA and AtNA (as well as control RNA) pools briefly to alkaline pH (50 mM NaOH at 65 °C (RNA (3 min), HNA/AtNA (27 min), neutralized with 1 M Tris•HCl pH 7.4) in order to generate an increased amount of >p products, before a prolonged incubation (−9 °C ice, 5 months). Using a novel engineered reverse transcriptase (RT TK², see Materials and methods), which, unlike commercial enzymes, is able to reverse transcribe both RNA as well as both HNA and AtNA oligomers, we analysed ligation products by RT-PCR. While HNA pools again proved inert, we now observed clear intermolecular recombination in the AtNA pools, although to a notably lesser extent than equivalent RNA pools.

Deep sequencing of the recombination products from both RNA and AtNA pools revealed a similar size distribution indicative of recombination but, in the case of RNA, biased towards smaller products, presumably due to increased hydrolytic degradation induced by exposure to alkaline pH (Figure 6C). Furthermore, a clear ApN nucleotide signature was observed at the AtNA recombination junction, distinct from the CpN signature of RNA, presumably reflecting the different chemical requirements for AtNA recombination (Figure 6D). The finding that only RNA (and to a lesser extent AtNA) pools are capable of intermolecular recombination, while DNA, ANA and HNA pools were inert, strongly suggests that a vicinal cis-diol configuration forms a critical chemical determinant of an innate intermolecular recombination potential with the propensity for recombination modulated by ring geometry.

Recombination is a fundamental process in biology both central to genetic diversification and genome repair (Pesce et al., 2016; de Visser and Elena, 2007), as well as a protective mechanism against the progressive corruption of genetic information by drift (i.e. Muller’s ratchet (Muller, 1964)). RNA recombination is widespread among RNA viruses (Simon-Loriere and Holmes, 2011); while generally attributed to template switching, recombination by purely chemical processes has been suggested to occur in polio (Gmyl et al., 1999) and rubella viruses (Adams et al., 2003), as well as Qbeta bacteriophage (Chetverin et al., 1997).

Here, we show that a capacity for spontaneous ligation and recombination is an innate property of random RNA (and to a lesser extent AtNA) oligomer pools and requires neither recombinase enzymes nor extraneous activating chemistry. As we demonstrate, such non-enzymatic recombination and ligation involves, to some degree, specific sequence motifs (e.g. in RNA pools) that promote ligation with either 2’−5’ or 3’−5’ regioselectivity (Figure 2, Figure 2—figure supplement 5). However, the bulk of observed reactivity derives from the spontaneous organization of random sequence oligomers into non-covalent complexes through intermolecular hybridization, which can be visualized by native gel electrophoresis (Figure 1—figure supplement 2).There is staggering potential combinatorial diversity of such intra-pool interactions. Within the N₂₀ pools examined herein (with a potential sequence diversity of ca. 10¹²), such combinatorial diversity amounts to >10²⁴ potential binary and an even vaster number of potential tertiary or higher order interactions. In the case of random RNA as well as semi-random RNA and AtNA oligomer pools, a subset of these non-covalent intermolecular assemblies do not remain inert (and non-covalent), but display a pervasive tendency to undergo spontaneous disproportionation reactions resulting in intermolecular recombination and ligation.

The discrepancy between the extent of ligation / recombination we observe (2–10 % of pool sequences (Figure 1, Figure 4) to the predicted frequency of functional sequences of, for example, aptamers / ribozymesfrom repertoire selection / SELEX experiments (1 in 10¹⁰-10¹³ (Wilson and Szostak, 1999)) should be viewed within the same context. Indeed, standard SELEX experiments are designed to iteratively deplete the interaction network's underlying pool reactivity and instead isolate the exceedingly rare unitary sequences capable of function. While such experiments have been ground-breaking in revealing the potential of RNA (as well as DNA and some XNAs) for complex catalytic or ligand-binding functions (Silverman, 2016; Taylor et al., 2015; Wilson and Szostak, 1999), our results show that a back-extrapolation from the hit frequency of SELEX experiments leads to a gross underestimation of global pool functionality, as only single sequences are considered and systems-level interactions and networks are ignored.

We were able to visualize the intermolecular recombination and / or ligation products both directly by gel shift in denaturing gel electrophoresis and by specific RT-PCR amplification. Both assays provide evidence for bona fide ligation and recombination products as PCR or sequencing artefacts can be ruled out on multiple grounds. For one, incubation at −80 °C, which freezes the eutectic phase in our buffer system (Mutschler et al., 2015; Mutschler and Holliger, 2014), never yields products, establishing that fragments do not recombine during workup. Furthermore, the appearance (or absence) of recombination products is clearly and reproducibly linked to chemical features of the original pool (e.g. free 5’OH (RNA) (Figure 4), or cis diol (RNA, AtNA) (Figure 6)), which are erased in the RT step. Finally, in both the case of the >p activated and unmodified random RNA sequence pools, we are able to observe the formation of ligation / recombination products directly by Urea-PAGE, which would dissociate even highly stable non-covalent complexes. Indeed, we note that our assays likely underestimate total pool reactivity (as well as the diversity of potential reaction products) as a range of more complex potential recombination modes, including intra- and intermolecular circular, lariat or branched products are only partially detected by our methods, or not at all (Figure 4—figure supplement 4).

Previous reports of non-enzymatic RNA recombination with defined substrates suggests that the majority of such reactions are likely to involve either a concerted mechanism of direct in-line attack on an internal phosphodiester linkage or, more frequently, a two step-mechanism of two consecutive transesterification reactions via an >p intermediate generated by hydrolysis (Lutay et al., 2007; Nechaev et al., 2009; Staroseletz et al., 2018). In both reaction trajectories, ligation /recombination is promoted by proximity through in trans intermolecular hybridization (Figure 4—figure supplement 1) as well as the concentration and entropic effects of eutectic ice phase formation. Whilst we observe formation of canonical 3’−5’ linkages with some RNA motifs (H4), we expect that the majority of RNA pool linkages likely conform to 2’−5’ regiochemistry as these linkages form preferentially in RNA duplex geometry (Lutay et al., 2006; Orgel, 2004), although such information is lacking for AtNA. In RNA, sporadic 2’−5’ linkages have been found to be broadly compatible with folding and function (Engelhart et al., 2013) and can progressively be converted to the canonical 3’−5’ linkage (Mariani and Sutherland, 2017).

The mechanistic basis for the distinctive sequence signature (CpN) in RNA is currently unclear, although CpA and UpA junctions have been shown to be generally more reactive (Kaukinen et al., 2002). Thus, the bias may reflect the higher propensity for C>p formation and/or reactivity, which may be more reactive due to the stronger electron withdrawal by the cytosine ring system (Krishnamurthy, 2012) or hydrogen bond interactions between the C-2 oxygen of the pyrimidine and the 2'-hydroxyl of the ribose (Bibillo et al., 2000). Earlier reports suggest that reactivity of phosphodiester bonds in a gapped duplex can be context dependent being strongly modulated by the local sequence (stacking) and base-pairing (H-bonding) (Kaukinen et al., 2002; Oivanen et al., 1998), which may explain the different AtNA recombination signature (ApN) (Figure 6D).

Repertoire selection experiments had previously shown that random sequence pools of different genetic polymers (including RNA, DNA and some XNAs) all contain functional sequences, including highly active ribozymes (Wachowius et al., 2017), DNAzymes (Silverman, 2016) and to a lesser extent ANA-, HNA- and other XNAzymes (Taylor et al., 2015). Although rare, these functional sequences can be isolated by iterative rounds of selection and amplification. In contrast, in the experiments presented here (which do not involve enrichment), we observe striking differences in innate reactive potential of different chemistries, with only RNA (and to a lesser extent AtNA) pools undergoing ligation and recombination. In contrast, DNA, ANA and HNA pools remained inert in our experiments, even under forcing conditions such as alkaline pH or high concentrations of transition metal ions like Zn²⁺, which have been shown to promote self-cleavage in DNA (Gu et al., 2013). While nucleophilicity and pK_a of the 5’-OH are likely to be comparable in RNA, DNA and the XNAs examined herein, classic studies using model nucleotide phosphodiester and -triester compounds suggest an at least 30-fold higher reactivity of RNA internucleotide diester linkages compared to DNA and ANA (Lönnberg and Korhonen, 2005). This increased reactivity is thought to be due to the vicinal cis-diol configuration of RNA, with the 2´-OH stabilizing the negative charge developing on the phosphorane intermediate and /or the departing 3´-oxygen by both H-bonding (Lönnberg and Korhonen, 2005; Oivanen et al., 1998) and local electronic effects (Aström et al., 2004). Consistent with this mechanistic model, AtNA, an RNA congener, sharing an analogous vicinal cis-diol configuration (in a six-membered ring context), was found to be capable of intermolecular recombination, although AtNA reactivity appears to be significantly lower than RNA. The latter may be due to greater torsional strain required for in-line attack or formation of an analogous cyclic phosphate (AtNA>p) intermediate (with an almost planar configuration) in the context of a six-membered ring (Bruzik et al., 1996).

Although >p is only weakly activated towards nucleophilic attack, in the context of high effective concentrations of a 5’OH nucleophile in a gapped duplex (or pre-organized internal loop motifs like H4, J4), intermolecular bond formation can be efficient (Usher and McHale, 1976; Lutay et al., 2006). Crucially, >p groups form spontaneously as part of RNA (and presumably AtNA) hydrolytic degradation. While DNA pools have been found to comprise a number of small self-cleaving motifs (Gu et al., 2013), these yield 3’OH and 5’p termini, which require further activation for ligation. Thus a vicinal diol configuration not only accelerates the initial strand cleavage step of recombination, but also yields cleavage product termini (>p and 5’OH) competent for re-ligation. Indeed, other vicinal cis-diol containing genetic polymers have previously been examined by solid-phase synthetic chemistry as part of systematic variation along the ribo- and lyxopyranosyl series (Eschenmoser, 1999; Wippo et al., 2001) (Figure 6—figure supplement 5), and ligative polymerization of 2’, 3’>p activated pyranosyl tetranucleotides on a template has been demonstrated (Bolli et al., 1997). Therefore, a vicinal diol configuration appears to be a critical component of a spontaneous capacity for recombination, setting RNA apart from a range of other prebiotically plausible genetic polymers including TNA (Schöning et al., 2000), ANA (Roberts et al., 2018) and PNA (Böhler et al., 1995).

The pervasive spontaneous ligation and recombination activity displayed by oligomer pools described herein may seem counterintuitive, but such behaviour is both predicted and supported by theoretical arguments from statistical mechanics and thermodynamics. These suggest that under the right boundary conditions, recombination - with an concomitant increase in the diversity of length distribution of (RNA) polymer chains - is favoured by entropic factors (Blokhuis and Lacoste, 2017). Remarkably, not only is the diversity in oligomer length distribution altered but recombined pools also display a shift towards an increased tendency for secondary structure formation (Figure 5E). This observation likely reflects both the tendency of longer oligomers to form more stable and more diverse secondary structures (Briones et al., 2009; Stich et al., 2008) as well as some degree of intrapool selection for sequences capable of intermolecular hybridization and duplex formation (Figure 1—figure supplement 2). Finally, recombination also scrambles parts of the pool sequence information, which could lead to an increase in informational complexity in redundant pools such as the random 20-mer RNA and semi-random 40-mer RNA and AtNA oligomer pools used in our experiments. To better understand this effect, we explored recombination outcomes by in silico simulations of a broad range of boundary conditions (including a range of hydrolytic cleavage (degradation) vs. ligation rates, pool redundancy and code complexity (2-letter vs. 4-letter code)). Using the Shannon population level entropy index D (Derr et al., 2012) as an established measure of informational complexity (see Material and methods), we find that recombination in simulated, redundant N₁₀ and N₂₀ pools disproportionates pool sequences into a distribution of both shorter (cleavage) and longer (ligation) strands and spontaneously increases D under a wide range of boundary conditions (Figure 7, Figure 7—figure supplement 1). Furthermore, in silico recombination reveals critical pool and reaction parameters that enhance information gains, whereby increases in pool informational complexity (as measured by D) scale with pool redundancy, code complexity and oligomer length (Figure 7—figure supplement 1). Under the conditions used in our experiments, i.e. ca. 1000-fold pool redundancy, a 4-letter base alphabet and N₂₀ oligomer length, information gains occur under almost all boundary conditions (Figure 7). Thus, spontaneous recombination in random oligomer pools under any but the most strongly degradative regimes expands both sequence size distribution and sequence diversity, and thereby progressively increases pool compositional (sequence length distribution), structural (secondary structure formation) and informational complexity.

Figure 7 with 1 supplement see all

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In the context of evolution of early random sequence oligomer pools (and potentially viral quasispecies), the impact of the above recombination processes is significant, in particular considering that such pools would at the same time likely to be under fractionation and/or adaptive pressure. Indeed, a number of physicochemical processes (including thermophoretic cycles in porous substrates (Kreysing et al., 2015) or adsorption to mineral surfaces (Ferris et al., 1996)) would favour length-extended oligomers over pool members or short cleavage products. However, adaptive pressure is arguably the most important process that favours more diverse and length extended oligomer sequences. Previous studies have underlined the importance of oligomer length for encoding the most active (large) functional motifs or more frequent smaller motifs (Ekland et al., 1995; Velez et al., 2012; Bartel and Szostak, 1993), as well as the importance of sequence diversity and the power of (continuous) recombination in accelerating adaptive walks (Stemmer, 1994; Hayden et al., 2005; Lehman, 2008; Arnold, 2009; Mutschler et al., 2015).

In conclusion, our findings indicate that native random-sequence RNA (and to a lesser extent AtNA) pools are naturally predisposed to recombination via near energetically neutral transesterification chemistry enabled by their specific chemical structure. As a result, such pools have an unique innate capacity to ‘bootstrap’ themselves towards higher compositional, informational and structural complexity (Briones et al., 2009) and thus a likely enhanced adaptive potential.

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for: Figure 1, Figure 1—figure supplement 1, Figure 2, Figure 2—figure supplement 1, Figure 4, Figure 4—figure supplement 2, Figure 4—figure supplement 3, Figure 5, Figure 5—figure supplement 1, Figure 6 and Figure 6—figure supplement 2. Further source files are shell scripts for the motif search, extraction of secondary structure frequencies and size distributions and python scripts for the generation of random sequences using position-specific nucleotide frequencies and the simulations of the population level Shannon Index at different cleavage / ligation rates are available online (see Source code 1-5).

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Author details

1. Hannes Mutschler

   MRC Laboratory of Molecular Biology, Cambridge, United Kingdom

   Present address

   Max Planck Institute of Biochemistry, Martinsried, Germany

   Contribution

   Conceptualization, Resources, Software, Formal analysis, Supervision, Validation, Investigation, Visualization, Methodology, Writing—original draft, Project administration, Writing—review and editing

   Contributed equally with

   Alexander I Taylor

   For correspondence

   mutschler@biochem.mpg.de

   Competing interests

   No competing interests declared

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

2. Alexander I Taylor

   MRC Laboratory of Molecular Biology, Cambridge, United Kingdom

   Contribution

   Conceptualization, Resources, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing

   Contributed equally with

   Hannes Mutschler

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7684-1437

3. Benjamin T Porebski

   MRC Laboratory of Molecular Biology, Cambridge, United Kingdom

   Contribution

   Software, Validation, Visualization, Methodology

   Competing interests

   No competing interests declared

4. Alice Lightowlers

   MRC Laboratory of Molecular Biology, Cambridge, United Kingdom

   Present address

   Weatherall Institute of Molecular Medicine, Department of Medicine, University of Oxford, Oxford, United Kingdom

   Contribution

   Validation, Investigation

   Competing interests

   No competing interests declared

5. Gillian Houlihan

   MRC Laboratory of Molecular Biology, Cambridge, United Kingdom

   Contribution

   Resources, Developed, produced and tested the new RT-enzyme

   Competing interests

   No competing interests declared

6. Mikhail Abramov

   REGA Institute, Katholieke Universiteit Leuven, Leuven, Belgium

   Contribution

   Resources, Synthesized and quality-checked the HNA and AtNA nucleosides used in this study

   Competing interests

   No competing interests declared

7. Piet Herdewijn

   REGA Institute, Katholieke Universiteit Leuven, Leuven, Belgium

   Contribution

   Resources, Funding acquisition

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-3589-8503

8. Philipp Holliger

   MRC Laboratory of Molecular Biology, Cambridge, United Kingdom

   Contribution

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

   For correspondence

   ph1@mrc-lmb.cam.ac.uk

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3440-9854

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