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The Tetrahymena ribozyme has been shown to catalyze an RNA polymerase-like reaction in which an RNA primer is extended by the sequential addition of pN nucleotides derived from GpN dinucleotides, where N = A, C, or U. Here, we show that this reaction is influenced by the presence of a template; bases that can form Watson-Crick base pairs with a template add as much as 25-fold more efficiently than mismatched bases. A mutant enzyme with an altered guanosine binding site can catalyze template-directed primer extension with all four bases when supplied with dinucleotides of the form 2-aminopurine-pN.

The group I self-splicing introns act at exon-intron junctions without recognizing a particular sequence. In order to understand splice-site selection, we have developed an assay system based on the Tetrahymena ribozyme to allow the study of numerous 5'-splice-site variants. Cleavage at the correct site requires formation of the correct secondary structure and occurs most efficiently within a 3-base-pair window centered on base pair 5 from the bottom of the P1 stem. Within this window the ribozyme recognizes and cleaves at a "wobble" base pair; the base pair above the cleavage site also influences splicing efficiency. The recognition of RNA structure rather than sequence explains the ability of these transposable introns to splice out of a variety of sequence contexts.

The self-splicing sunY intron from bacteriophage T4 has the smallest conserved core secondary structure of any of the active group I introns. Here we show that several nonconserved regions can be deleted from this intron without complete loss of catalytic activity. The 3' stems P9, P9.1, and P9.2 can be deleted while retaining 5' cleaving activity. Two base-paired stems (P7.1 and P7.2) that are peculiar to the group IA introns can also be deleted; however, the activities of the resulting derivatives depend greatly on the choice of replacement sequences and their lengths. The smallest active derivative is less than 180 nucleotides long. These experiments help to define the minimum structural requirements for catalysis.

The prebiotic formation of RNA building blocks is well-supported experimentally, yet the emergence of sequence- and structure-specific RNA oligomers is generally attributed to biological selection via Darwinian evolution rather than prebiotic chemical selectivity. In this study, we used deep sequencing to investigate the partitioning of randomized RNA overhangs into ligated products by either splinted ligation or loop-closing ligation. Comprehensive sequence-reactivity profiles revealed that loop-closing ligation preferentially yields hairpin structures with loop sequences UNNG, CNNG, and GNNA (where N represents A, C, G, or U) under competing conditions. In contrast, splinted ligation products tended to be GC rich. Notably, the overhang sequences that preferentially partition to loop-closing ligation significantly overlap with the most common biological tetraloops, whereas the overhangs favoring splinted ligation exhibit an inverse correlation with biological tetraloops. Applying these sequence rules enables the high-efficiency assembly of functional ribozymes from short RNAs without template inhibition. Our findings suggest that the RNA tetraloop structures that are common in biology may have been predisposed and prevalent in the prebiotic pool of RNAs, prior to the advent of Darwinian evolution. We suggest that the one-step prebiotic chemical process of loop-closing ligation could have favored the emergence of the first RNA functions., (© 2024 The Author(s). Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Broad discussion is needed to chart a path forward.

The RNA World hypothesis posits that RNA was the molecule of both heredity and function during the emergence of life. This hypothesis implies that RNA templates can be copied, and ultimately replicated, without the catalytic aid of evolved enzymes. A major problem with nonenzymatic template-directed polymerization has been the very poor copying of sequences containing rA and rU. Here, we overcome that problem by using a prebiotically plausible mixture of RNA mononucleotides and random-sequence oligonucleotides, all activated by methyl isocyanide chemistry, that direct the uniform copying of arbitrary-sequence templates, including those harboring rA and rU. We further show that the use of this mixture in copying reactions suppresses copying errors while also generating a more uniform distribution of mismatches than observed for simpler systems. We find that oligonucleotide competition for template binding sites, oligonucleotide ligation and the template binding properties of reactant intermediates work together to reduce product sequence bias and errors. Finally, we show that iterative cycling of templated polymerization and activation chemistry improves the yields of random-sequence products. These results for random-sequence template copying are a significant advance in the pursuit of nonenzymatic RNA replication., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Transfer RNAs (tRNA) are decorated during biogenesis with a variety of modifications that modulate their stability, aminoacylation, and decoding potential during translation. The complex landscape of tRNA modification presents significant analysis challenges and to date no single approach enables the simultaneous measurement of important but disparate chemical properties of individual, mature tRNA molecules. We developed a new, integrated approach to analyze the sequence, modification, and aminoacylation state of tRNA molecules in a high throughput nanopore sequencing experiment, leveraging a chemical ligation that embeds the charged amino acid in an adapted tRNA molecule. During nanopore sequencing, the embedded amino acid generates unique distortions in ionic current and translocation speed, enabling application of machine learning approaches to classify charging status and amino acid identity. Specific applications of the method indicate it will be broadly useful for examining relationships and dependencies between tRNA sequence, modification, and aminoacylation., Competing Interests: Conflict of interest statement A.R., L.K.W., J.W.S, and J.R.H. are authors on a provisional patent covering this technology. L.K.W. has received travel and accommodation expenses from Oxford Nanopore Technologies to present at scientific meetings, and was a participant in the ONT Early Access program for SQK-RNA004.

The acquisition of new RNA functions through evolutionary processes was essential for the diversification of RNA-based primordial biology and its subsequent transition to modern biology. However, the mechanisms by which RNAs access new functions remain unclear. Do RNA enzymes need completely new folds to support new but related functions, or is reoptimization of the active site sufficient? What are the roles of neutral and adaptive mutations in evolutionary innovation? Here, we address these questions experimentally by focusing on the evolution of substrate specificity in RNA-catalyzed RNA assembly. We use directed in vitro evolution to show that a ligase ribozyme that uses prebiotically relevant 5'-phosphorimidazole-activated substrates can be evolved to catalyze ligation with substrates that are 5'-activated with the biologically relevant triphosphate group. Interestingly, despite catalyzing a related reaction, the new ribozyme folds into a completely new structure and exhibits promiscuity by catalyzing RNA ligation with both triphosphate and phosphorimidazole-activated substrates. Although distinct in sequence and structure, the parent phosphorimidazolide ligase and the evolved triphosphate ligase ribozymes can be connected by a series of point mutations where the intermediate sequences retain at least some ligase activity. The existence of a quasi-neutral pathway between these distinct ligase ribozymes suggests that neutral drift is sufficient to enable the acquisition of new substrate specificity, thereby providing opportunities for subsequent adaptive optimization. The transition from RNA-catalyzed RNA assembly using phosphorimidazole-activated substrates to triphosphate-activated substrates may have foreshadowed the later evolution of the protein enzymes that use monomeric triphosphates (nucleoside triphosphates, NTPs) for RNA synthesis., Competing Interests: Competing interests statement:The authors declare no competing interest.

Coded ribosomal peptide synthesis could not have evolved unless its sequence and amino acid-specific aminoacylated tRNA substrates already existed. We therefore wondered whether aminoacylated RNAs might have served some primordial function prior to their role in protein synthesis. Here, we show that specific RNA sequences can be nonenzymatically aminoacylated and ligated to produce amino acid-bridged stem-loop RNAs. We used deep sequencing to identify RNAs that undergo highly efficient glycine aminoacylation followed by loop-closing ligation. The crystal structure of one such glycine-bridged RNA hairpin reveals a compact internally stabilized structure with the same eponymous T-loop architecture that is found in many noncoding RNAs, including the modern tRNA. We demonstrate that the T-loop-assisted amino acid bridging of RNA oligonucleotides enables the rapid template-free assembly of a chimeric version of an aminoacyl-RNA synthetase ribozyme. We suggest that the primordial assembly of amino acid-bridged chimeric ribozymes provides a direct and facile route for the covalent incorporation of amino acids into RNA. A greater functionality of covalently incorporated amino acids could contribute to enhanced ribozyme catalysis, providing a driving force for the evolution of sequence and amino acid-specific aminoacyl-RNA synthetase ribozymes in the RNA World. The synthesis of specifically aminoacylated RNAs, an unlikely prospect for nonenzymatic reactions but a likely one for ribozymes, could have set the stage for the subsequent evolution of coded protein synthesis., Competing Interests: Competing interests statement:The authors declare no competing interest.

Membraneless coacervate microdroplets have long been proposed as model protocells as they can grow, divide, and concentrate RNA by natural partitioning. However, the rapid exchange of RNA between these compartments, along with their rapid fusion, both within minutes, means that individual droplets would be unable to maintain their separate genetic identities. Hence, Darwinian evolution would not be possible, and the population would be vulnerable to collapse due to the rapid spread of parasitic RNAs. In this study, we show that distilled water, mimicking rain/freshwater, leads to the formation of electrostatic crosslinks on the interface of coacervate droplets that not only suppress droplet fusion indefinitely but also allow the spatiotemporal compartmentalization of RNA on a timescale of days depending on the length and structure of RNA. We suggest that these nonfusing membraneless droplets could potentially act as protocells with the capacity to evolve compartmentalized ribozymes in prebiotic environments.

Extant life contains numerous non-equilibrium mechanisms to create order not achievable at equilibrium; it is generally assumed that these mechanisms evolved because the resulting order was sufficiently beneficial to overcome associated costs of time and energy. Here, we identify a broad range of conditions under which non-equilibrium order-creating mechanisms will evolve as an inevitable consequence of self-replication, even if the order is not directly functional. We show that models of polymerases, when expanded to include known stalling effects, can evolve kinetic proofreading through selection for fast replication alone, consistent with data from recent mutational screens. Similarly, replication contingent on fast self-assembly can select for non-equilibrium instabilities and result in more ordered structures without any direct selection for order. We abstract these results into a framework that predicts that self-replication intrinsically amplifies dissipative order-enhancing mechanisms if the distribution of replication times is wide enough. Our work suggests the intriguing possibility that non-equilibrium order can arise more easily than assumed, even before that order is directly functional, with consequences impacting such diverse phenomena as the evolution of mutation rates, kinetic traps in self-assembly, and the origin of life.

Multiple RNA strands can interact in solution and assume a large variety of configurations dictated by their potential for base pairing. Although duplex formation from two complementary oligonucleotides has been studied in detail, we still lack a systematic characterization of the behavior of higher order complexes. Here, we focus on the thermodynamic and kinetic effects of an upstream oligonucleotide on the binding of a downstream oligonucleotide to a common template, as we vary the sequence and structure of the contact interface. We show that coaxial stacking in RNA is well correlated with but much more stabilizing than helix propagation over an analogous intact double helix step (median ΔΔ G ° 37 °C ≈ 1.7 kcal/mol). Consequently, approximating coaxial stacking in RNA with the helix propagation term leads to large discrepancies between predictions and our experimentally determined melting temperatures, with an offset of ≈10 °C. Our kinetic study reveals that the hybridization of the downstream probe oligonucleotide is impaired (lower k on ) by the presence of the upstream oligonucleotide, with the thermodynamic stabilization coming entirely from an extended lifetime (lower k off ) of the bound downstream oligonucleotide, which can increase from seconds to months. Surprisingly, we show that the effect of nicks is dependent on the length of the stacking oligonucleotides, and we discuss the binding of ultrashort (1-4 nt) oligonucleotides that are relevant in the context of the origin of life. The thermodynamic and kinetic data obtained in this work allow for the prediction of the formation and stability of higher-order multistranded complexes.

The mechanism by which genetic information was copied prior to the evolution of ribozymes is of great interest because of its importance to the origin of life. The most effective known process for the nonenzymatic copying of an RNA template is primer extension by a two-step pathway in which 2-aminoimidazole-activated nucleotides first react with each other to form an imidazolium-bridged intermediate that subsequently reacts with the primer. Reaction kinetics, structure-activity relationships, and X-ray crystallography have provided insight into the overall reaction mechanism, but many puzzles remain. In particular, high concentrations of Mg 2+ are required for efficient primer extension, but the mechanism by which Mg 2+ accelerates primer extension remains unknown. By analogy with the mechanism of DNA and RNA polymerases, a role for Mg 2+ in facilitating the deprotonation of the primer 3'-hydroxyl is often assumed, but no catalytic metal ion is seen in crystal structures of the primer-extension complex. To explore the potential effects of Mg 2+ binding in the reaction center, we performed atomistic molecular dynamics simulations of a series of modeled complexes in which a Mg 2+ ion was placed in the reaction center with inner-sphere coordination with different sets of functional groups. Our simulations suggest that coordination of a Mg 2+ ion with both O3' of the terminal primer nucleotide and the pro-S p nonbridging oxygen of the reactive phosphate of an imidazolium-bridged dinucleotide would help to pre-organize the structure of the primer/template substrate complex to favor the primer-extension reaction. Our results suggest that the catalytic metal ion may play an important role in overcoming electrostatic repulsion between a deprotonated O3' and the reactive phosphate of the bridged dinucleotide and lead to testable predictions of the mode of Mg 2+ binding that is most relevant to catalysis of primer extension., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

In the RNA World before the emergence of an RNA polymerase, nonenzymatic template copying would have been essential for the transmission of genetic information. However, the products of chemical copying with the canonical nucleotides (A, U, C, and G) are heavily biased toward the incorporation of G and C, which form a more stable base pair than A and U. We therefore asked whether replacing adenine (A) with diaminopurine (D) might lead to more efficient and less biased nonenzymatic template copying by making a stronger version of the A:U pair. As expected, primer extension substrates containing D bound to U in the template more tightly than substrates containing A. However, primer extension with D exhibited elevated reaction rates on a C template, leading to concerns about fidelity. Our crystallographic studies revealed the nature of the D:C mismatch by showing that D can form a wobble-type base pair with C. We then asked whether competition with G would decrease the mismatched primer extension. We performed nonenzymatic primer extension with all four activated nucleotides on randomized RNA templates containing all four letters and used deep sequencing to analyze the products. We found that the DUCG genetic system exhibited a more even product distribution and a lower mismatch frequency than the canonical AUCG system. Furthermore, primer extension is greatly reduced following all mismatches, including the D:C mismatch. Our study suggests that D deserves further attention for its possible role in the RNA World and as a potentially useful component of artificial nonenzymatic RNA replication systems.

The emergence of RNA on the early Earth is likely to have been influenced by chemical and physical processes that acted to filter out various alternative nucleic acids. For example, UV photostability is thought to have favored the survival of the canonical nucleotides. In a recent proposal for the prebiotic synthesis of the building blocks of RNA, ribonucleotides share a common pathway with arabino- and threo-nucleotides. We have therefore investigated non-templated primer extension with 2-aminoimidazole-activated forms of these alternative nucleotides to see if the synthesis of the first oligonucleotides might have been biased in favor of RNA. We show that non-templated primer extension occurs predominantly through 5'-5' imidazolium-bridged dinucleotides, echoing the mechanism of template-directed primer extension. Ribo- and arabino-nucleotides exhibited comparable rates and yields of non-templated primer extension, whereas threo-nucleotides showed lower reactivity. Competition experiments confirmed the bias against the incorporation of threo-nucleotides. The incorporation of an arabino-nucleotide at the end of the primer acts as a chain terminator and blocks subsequent extension. These biases, coupled with potentially selective prebiotic synthesis, and the templated copying that is known to favour the incorporation of ribonucleotides, provide a plausible model for the effective exclusion of arabino- and threo-nucleotides from primordial oligonucleotides., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Nonenzymatic template-directed RNA copying requires catalysis by divalent metal ions. The primer extension reaction involves the attack of the primer 3'-hydroxyl on the adjacent phosphate of a 5'-5'-imidazolium-bridged dinucleotide substrate. However, the nature of the interaction of the catalytic metal ion with the reaction center remains unclear. To explore the coordination of the catalytic metal ion with the imidazolium-bridged dinucleotide substrate, we examined catalysis by oxophilic and thiophilic metal ions with both diastereomers of phosphorothioate-modified substrates. We show that Mg 2+ and Cd 2+ exhibit opposite preferences for the two phosphorothioate substrate diastereomers, indicating a stereospecific interaction of the divalent cation with one of the nonbridging phosphorus substituents. High-resolution X-ray crystal structures of the products of primer extension with phosphorothioate substrates reveal the absolute stereochemistry of this interaction and indicate that catalysis by Mg 2+ involves inner-sphere coordination with the nonbridging phosphate oxygen in the pro- S P position, while thiophilic cadmium ions interact with sulfur in the same position, as in one of the two phosphorothioate substrates. These results collectively suggest that during nonenzymatic RNA primer extension with a 5'-5'-imidazolium-bridged dinucleotide substrate the interaction of the catalytic Mg 2+ ion with the pro- S P oxygen of the reactive phosphate plays a crucial role in the metal-catalyzed S N 2(P) reaction.

Oligonucleotide hybridization is crucial in various biological, prebiotic and nanotechnological processes, including gene regulation, non-enzymatic primer extension and DNA nanodevice assembly. Although extensive research has focused on the thermodynamics and kinetics of nucleic acid hybridization, the behavior of complex mixtures and the outcome of competition for target binding remain less well understood. In this study, we investigate the impact of mismatches and bulges in a 12 bp DNA or RNA duplex on its association (kon) and dissociation (koff) kinetics. We find that such defects have relatively small effects on the association kinetics, while the dissociation kinetics vary in a position-dependent manner by up to 6 orders of magnitude. Building upon this observation, we explored a competition scenario involving multiple oligonucleotides, and observed a transient low specificity of probe hybridization to fully versus partially complementary targets in solution. We characterize these long-lived metastable states and their evolution toward equilibrium, and show that sufficiently long-lived mis-paired duplexes can serve as substrates for prebiotically relevant chemical copying reactions. Our results suggest that transient low accuracy states may spontaneously emerge within all complex nucleic acid systems comprising a large enough number of competing strands, with potential repercussions for gene regulation in the realm of modern biology and the prebiotic preservation of genetic information., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The origin of life likely occurred within environments that concentrated cellular precursors and enabled their co-assembly into cells. Soda lakes (those dominated by Na + ions and carbonate species) can concentrate precursors of RNA and membranes, such as phosphate, cyanide, and fatty acids. Subsequent assembly of RNA and membranes into cells is a long-standing problem because RNA function requires divalent cations, e.g. Mg 2+ , but Mg 2+ disrupts fatty acid membranes. The low solubility of Mg-containing carbonates limits soda lakes to moderate Mg 2+ concentrations (∼1 mM), so we investigated whether both RNAs and membranes function within these lakes. We collected water from Last Chance Lake and Goodenough Lake in Canada. Because we sampled after seasonal evaporation, the lake water contained ∼1 M Na + and ∼1 mM Mg 2+ near pH 10. In the laboratory, nonenzymatic, RNA-templated polymerization of 2-aminoimidazole-activated ribonucleotides occurred at comparable rates in lake water and standard laboratory conditions (50 mM MgCl 2 , pH 8). Additionally, we found that a ligase ribozyme that uses oligonucleotide substrates activated with 2-aminoimidazole was active in lake water after adjusting pH from ∼10 to 9. We also observed that decanoic acid and decanol assembled into vesicles in a dilute solution that resembled lake water after seasonal rains, and that those vesicles retained encapsulated solutes despite salt-induced flocculation when the external solution was replaced with dry-season lake water. By identifying compatible conditions for nonenzymatic and ribozyme-catalyzed RNA assembly, and for encapsulation by membranes, our results suggest that soda lakes could have enabled cellular life to emerge on Earth, and perhaps elsewhere., (© The Author(s) 2024. Published by Oxford University Press on behalf of National Academy of Sciences.)

Coded ribosomal peptide synthesis could not have evolved unless its sequence and amino acid specific aminoacylated tRNA substrates already existed. We therefore wondered whether aminoacylated RNAs might have served some primordial function prior to their role in protein synthesis. Here we show that specific RNA sequences can be nonenzymatically aminoacylated and ligated to produce amino acid-bridged stem-loop RNAs. We used deep sequencing to identify RNAs that undergo highly efficient glycine aminoacylation followed by loop-closing ligation. The crystal structure of one such glycine-bridged RNA hairpin reveals a compact internally stabilized structure with the same eponymous T-loop architecture found in modern tRNA. We demonstrate that the T-loop assisted amino acid bridging of RNA oligonucleotides enables the rapid template-free assembly of a chimeric version of an aminoacyl-RNA synthetase ribozyme. We suggest that the primordial assembly of such chimeric ribozymes would have allowed the greater functionality of amino acids to contribute to enhanced ribozyme catalysis, providing a driving force for the evolution of sequence and amino acid specific aminoacyl-RNA synthetase enzymes prior to their role in protein synthesis.

2-Thiouridine (s 2 U) is a nucleobase modification that confers enhanced efficiency and fidelity both on modern tRNA codon translation and on nonenzymatic and ribozyme-catalyzed RNA copying. We have discovered an unusual base pair between two 2-thiouridines that stabilizes an RNA duplex to a degree that is comparable to that of a native A:U base pair. High-resolution crystal structures indicate similar base-pairing geometry and stacking interactions in duplexes containing s 2 U:s 2 U compared to those with U:U pairs. Notably, the C═O···H-N hydrogen bond in the U:U pair is replaced with a C═S···H-N hydrogen bond in the s 2 U:s 2 U base pair. The thermodynamic stability of the s 2 U:s 2 U base pair suggested that this self-pairing might lead to an increased error frequency during nonenzymatic RNA copying. However, competition experiments show that s 2 U:s 2 U base-pairing induces only a low level of misincorporation during nonenzymatic RNA template copying because the correct A:s 2 U base pair outcompetes the slightly weaker s 2 U:s 2 U base pair. In addition, even if an s 2 U is incorrectly incorporated, the addition of the next base is greatly hindered. This strong stalling effect would further increase the effective fidelity of nonenzymatic RNA copying with s 2 U. Our findings suggest that s 2 U may enhance the rate and extent of nonenzymatic copying with only a minimal cost in fidelity.

Charge separation is one of the most common consequences of the absorption of UV light by DNA. Recently, it has been shown that this process can enable efficient self-repair of cyclobutane pyrimidine dimers (CPDs) in specific short DNA oligomers such as the GAT[double bond, length as m-dash]T sequence. The mechanism was characterized as sequential electron transfer through the nucleobase stack which is controlled by the redox potentials of nucleobases and their sequence. Here, we demonstrate that the inverse sequence T[double bond, length as m-dash]TAG promotes self-repair with higher quantum yields (0.58 ± 0.23%) than GAT[double bond, length as m-dash]T (0.44 ± 0.18%) in a comparative study involving UV-irradiation experiments. After extended exposure to UV irradiation, a photostationary equilibrium between self-repair and damage formation is reached at 33 ± 13% for GAT[double bond, length as m-dash]T and at 40 ± 16% for T[double bond, length as m-dash]TAG, which corresponds to the maximum total yield of self-repair. Molecular dynamics and quantum mechanics/molecular mechanics (QM/MM) simulations allowed us to assign this disparity to better stacking overlap between the G and A bases, which lowers the energies of the key A - ˙G + ˙ charge transfer state in the dominant conformers of the T[double bond, length as m-dash]TAG tetramer. These conformational differences also hinder alternative photorelaxation pathways of the T[double bond, length as m-dash]TAG tetranucleotide, which otherwise compete with the sequential electron transfer mechanism responsible for CPD self-repair. Overall, we demonstrate that photoinduced electron transfer is strongly dependent on conformation and the availability of alternative photodeactivation mechanisms. This knowledge can be used in the identification and prediction of canonical and modified DNA sequences exhibiting efficient electron transfer. It also further contributes to our understanding of DNA self-repair and its potential role in the photochemical selection of the most photostable sequences on the early Earth., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

There is currently no plausible path for the emergence of a self-replicating protocell, because prevalent formulations of model protocells are built with fatty acid vesicles that cannot withstand the concentrations of Mg 2+ needed for the function and replication of nucleic acids. Although prebiotic chelates increase the survivability of fatty acid vesicles, the resulting model protocells are incapable of growth and division. Here, we show that protocells made of mixtures of cyclophospholipids and fatty acids can grow and divide in the presence of Mg 2+ -citrate. Importantly, these protocells retain encapsulated nucleic acids during growth and division, can acquire nucleotides from their surroundings, and are compatible with the nonenzymatic extension of an RNA oligonucleotide, chemistry needed for the replication of a primitive genome. Our work shows that prebiotically plausible mixtures of lipids form protocells that are active under the conditions necessary for the emergence of Darwinian evolution.

Vesicle fusion is an important process underlying cell division, transport, and membrane trafficking. In phospholipid systems, a range of fusogens including divalent cations and depletants have been shown to induce adhesion, hemifusion, and then full content fusion between vesicles. This work shows that these fusogens do not perform the same function for fatty acid vesicles, which are used as model protocells (primitive cells). Even when fatty acid vesicles appear adhered or hemifused to each other, the intervening barriers between vesicles do not rupture. This difference is likely because fatty acids have a single aliphatic tail, and are more dynamic than their phospholipid counterparts. To address this, it is postulated that fusion could instead occur under conditions, such as lipid exchange, that disrupt lipid packing. Using both experiments and molecular dynamics simulations, it is verified that fusion in fatty acid systems can indeed be induced by lipid exchange. These results begin to probe how membrane biophysics could constrain the evolutionary dynamics of protocells., (© 2023 The Authors. Small Methods published by Wiley-VCH GmbH.)

Nucleic acids can be damaged by ultraviolet (UV) irradiation, forming structural photolesions such as cyclobutane-pyrimidine-dimers (CPD). In modern organisms, sophisticated enzymes repair CPD lesions in DNA, but to our knowledge, no RNA-specific enzymes exist for CPD repair. Here, we show for the first time that RNA can protect itself from photolesions by an intrinsic UV-induced self-repair mechanism. This mechanism, prior to this study, has exclusively been observed in DNA and is based on charge transfer from CPD-adjacent bases. In a comparative study, we determined the quantum yields of the self-repair of the CPD-containing RNA sequence, GAU = U to GAUU (0.23%), and DNA sequence, d(GAT = T) to d(GATT) (0.44%), upon 285 nm irradiation via UV/Vis spectroscopy and HPLC analysis. After several hours of irradiation, a maximum conversion yield of ∼16% for GAU = U and ∼33% for d(GAT = T) was reached. We examined the dynamics of the intermediate charge transfer (CT) state responsible for the self-repair with ultrafast UV pump - IR probe spectroscopy. In the dinucleotides GA and d(GA), we found comparable quantum yields of the CT state of ∼50% and lifetimes on the order of several hundred picoseconds. Charge transfer in RNA strands might lead to reactions currently not considered in RNA photochemistry and may help understanding RNA damage formation and repair in modern organisms and viruses. On the UV-rich surface of the early Earth, these self-stabilizing mechanisms likely affected the selection of the earliest nucleotide sequences from which the first organisms may have developed.

A DNA polymerase with a single mutation and a divalent calcium cofactor catalyzes the synthesis of unnatural N3'→P5' phosphoramidate (NP) bonds to form NP-DNA. However, this template-directed phosphoryl transfer activity remains orders of magnitude slower than native phosphodiester synthesis. Here, we used time-resolved x-ray crystallography to show that NP-DNA synthesis proceeds with a single detectable calcium ion in the active site. Using insights from isotopic and elemental effects, we propose that one-metal-ion electrophilic substrate activation is inferior to the native two-metal-ion mechanism. We found that this deficiency in divalent activation could be ameliorated by trivalent rare earth and post-transition metal cations, substantially enhancing NP-DNA synthesis. Scandium(III), in particular, confers highly specific NP activity with kinetics enhanced by more than 100-fold over calcium(II), yielding NP-DNA strands up to 100 nucleotides in length.

Hybridization of short nucleic acid segments (<4 nt) to single-strand templates occurs as a critical intermediate in processes such as nonenzymatic nucleic acid replication and toehold-mediated strand displacement. These templates often contain adjacent duplex segments that stabilize base pairing with single-strand gaps or overhangs, but the thermodynamics and kinetics of hybridization in such contexts are poorly understood because of the experimental challenges of probing weak binding and rapid structural dynamics. Here we develop an approach to directly measure the thermodynamics and kinetics of DNA and RNA dinucleotide dehybridization using steady-state and temperature-jump infrared spectroscopy. Our results suggest that dinucleotide binding is stabilized through coaxial stacking interactions with the adjacent duplex segments as well as from potential noncanonical base-pairing configurations and structural dynamics of gap and overhang templates revealed using molecular dynamics simulations. We measure timescales for dissociation ranging from 0.2-40 μs depending on the template and temperature. Dinucleotide hybridization and dehybridization involve a significant free energy barrier with characteristics resembling that of canonical oligonucleotides. Together, our work provides an initial step for predicting the stability and kinetics of hybridization between short nucleic acid segments and various templates., Competing Interests: Declaration of interests A.L.F. is a co-founder and consultant of Evozyne, Inc. and a co-author of US Patent Applications 16/887,710 and 17/642,582; US Provisional Patent Applications 62/853,919, 62/900,420, 63/314,898, and 63/479,378; and International Patent Applications PCT/US2020/035206 and PCT/US2020/050466., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Catalytic RNAs or ribozymes are considered to be central to primordial biology. Most ribozymes require moderate to high concentrations of divalent cations such as Mg 2+ to fold into their catalytically competent structures and perform catalysis. However, undesirable effects of Mg 2+ such as hydrolysis of reactive RNA building blocks and degradation of RNA structures are likely to undermine its beneficial roles in ribozyme catalysis. Further, prebiotic cell-like compartments bounded by fatty acid membranes are destabilized in the presence of Mg 2+ , making ribozyme function inside prebiotically relevant protocells a significant challenge. Therefore, we sought to identify conditions that would enable ribozymes to retain activity at low concentrations of Mg 2+ . Inspired by the ability of ribozymes to function inside crowded cellular environments with <1 mM free Mg 2+ , we tested molecular crowding as a potential mechanism to lower the Mg 2+ concentration required for ribozyme-catalyzed RNA assembly. Here, we show that the ribozyme-catalyzed ligation of phosphorimidazolide RNA substrates is significantly enhanced in the presence of the artificial crowding agent polyethylene glycol. We also found that molecular crowding preserves ligase activity under denaturing conditions such as alkaline pH and the presence of urea. Additionally, we show that crowding-induced stimulation of RNA-catalyzed RNA assembly is not limited to phosphorimidazolide ligation but extends to the RNA-catalyzed polymerization of nucleoside triphosphates. RNA-catalyzed RNA ligation is also stimulated by the presence of prebiotically relevant small molecules such as ethylene glycol, ribose, and amino acids, consistent with a role for molecular crowding in primordial ribozyme function and more generally in the emergence of RNA-based cellular life., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

Demonstrating RNA catalysis within prebiotically relevant models of primordial cells (protocells) remains a challenge in origins of life research. Fatty acid vesicles encapsulating genomic and catalytic RNAs (ribozymes) are attractive models for protocells; however, RNA catalysis has largely been incompatible with fatty acid vesicles due to their instability in the presence of Mg 2+ at the concentrations required for ribozyme function. Here, we report a ribozyme that catalyzes template-directed RNA ligation at low Mg 2+ concentrations and thus remains active within stable vesicles. Ribose and adenine, both prebiotically relevant molecules, were found to greatly reduce Mg 2+ -induced RNA leakage from vesicles. When we co-encapsulated the ribozyme, substrate, and template within fatty acid vesicles, we observed efficient RNA-catalyzed RNA ligation upon subsequent addition of Mg 2+ . Our work shows that RNA-catalyzed RNA assembly can occur efficiently within prebiotically plausible fatty acid vesicles and represents a step toward the replication of primordial genomes within self-replicating protocells., (© 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

A key challenge in origin-of-life research is the identification of plausible conditions that facilitate multiple steps along the pathway from chemistry to biology. The incompatibility of nucleotide activation chemistry and nonenzymatic template-directed RNA copying has hindered attempts to define such a pathway. Here, we show that adding heteroaromatic small molecules to the reaction network facilitates in situ nucleotide phosphate activation under conditions compatible with RNA copying, allowing both reactions to take place in the same mixture. This is achieved using Passerini-type phosphate activation in concert with nucleophilic organocatalysts that intercept high-energy reactive intermediates; this sequence ultimately affords 5',5'-imidazolium-bridged dinucleotides─the active species in template-directed RNA polymerization. Our results suggest that mixtures of prebiotically relevant heteroaromatic small molecules could have played a key role in the transition from chemistry to biology.

The nonenzymatic copying of RNA is thought to have been necessary for the transition between prebiotic chemistry and ribozyme-catalyzed RNA replication in the RNA World. We have previously shown that a potentially prebiotic nucleotide activation pathway based on phospho-Passerini chemistry can lead to the efficient synthesis of 2-aminoimidazole activated mononucleotides when carried out under freeze-thaw cycling conditions. Such activated nucleotides react with each other to form 5'-5' 2-aminoimidazolium bridged dinucleotides, enabling template-directed primer extension to occur within the same reaction mixture. However, mononucleotides linked to oligonucleotides by a 5'-5' 2-aminoimidazolium bridge are superior substrates for nonenzymatic primer extension; their higher intrinsic reactivity and their higher template affinity enable faster template copying at lower substrate concentrations. Here we show that eutectic phase phospho-Passerini chemistry efficiently activates short oligonucleotides and promotes the formation of monomer-bridged-oligonucleotide species during freeze-thaw cycles. We then demonstrate that in-situ generated monomer-bridged-oligonucleotides lead to efficient nonenzymatic template copying in the same reaction mixture. Our demonstration that multiple steps in the pathway from activation chemistry to RNA copying can occur together in a single complex environment simplifies this aspect of the origin of life., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Semipermeable membranes are a key feature of all living organisms. While specialized membrane transporters in cells can import otherwise impermeable nutrients, the earliest cells would have lacked a mechanism to import nutrients rapidly under nutrient-rich circumstances. Using both experiments and simulations, we find that a process akin to passive endocytosis can be recreated in model primitive cells. Molecules that are too impermeable to be absorbed can be taken up in a matter of seconds in an endocytic vesicle. The internalized cargo can then be slowly released over hours, into the main lumen or putative cytoplasm. This work demonstrates a way by which primitive life could have broken the symmetry of passive permeation prior to the evolution of protein transporters.

Hybridization of short nucleic acid segments (<4 nucleotides) to single-strand templates occurs as a critical intermediate in processes such as non-enzymatic nucleic acid replication and toehold-mediated strand displacement. These templates often contain adjacent duplex segments that stabilize base pairing with single-strand gaps or overhangs, but the thermodynamics and kinetics of hybridization in such contexts are poorly understood due to experimental challenges of probing weak binding and rapid structural dynamics. Here we develop an approach to directly measure the thermodynamics and kinetics of DNA and RNA dinucleotide dehybridization using steady-state and temperature-jump infrared spectroscopy. Our results suggest that dinucleotide binding is stabilized through coaxial stacking interactions with the adjacent duplex segments as well as from potential non-canonical base pairing configurations and structural dynamics of gap and overhang templates revealed using molecular dynamics simulations. We measure timescales for dissociation ranging from 0.2 to 40 µs depending on the template and temperature. Dinucleotide hybridization and dehybridization involves a significant free energy barrier with characteristics resembling that of canonical oligonucleotides. Together, our work provides an initial step for predicting the stability and kinetics of hybridization between short nucleic acid segments and various templates.

The virtual circular genome (VCG) model was proposed as a means of going beyond template copying to indefinite cycles of nonenzymatic RNA replication during the origin of life. In the VCG model, the protocellular genome is a collection of short oligonucleotides that map to both strands of a virtual circular sequence. Replication is driven by templated nonenzymatic primer extensions on a subset of kinetically trapped partially base-paired configurations, followed by the shuffling of these configurations to enable continued oligonucleotide elongation. Here, we describe initial experimental studies of the feasibility of the VCG model for replication. We designed a small 12-nucleotide model VCG and synthesized all 247 oligonucleotides of lengths 2 to 12 corresponding to this genome. We experimentally monitored the fate of individual labeled primers in the pool of VCG oligonucleotides following the addition of activated nucleotides and investigated the effect of factors such as oligonucleotide length, concentration, composition, and temperature on the extent of primer extension. We observe a surprisingly prolonged equilibration process in the VCG system that enables a considerable extent of reaction. We find that environmental fluctuations would be essential for continuous templated extension of the entire VCG system since the shortest oligonucleotides can only bind to templates at low temperatures, while the longest oligonucleotides require high-temperature spikes to escape from inactive configurations. Finally, we demonstrate that primer extension is significantly enhanced when the mix of VCG oligonucleotides is preactivated. We discuss the necessity of ongoing in situ activation chemistry for continuous and accurate VCG replication.

In all known genetic polymers, molecular recognition via hydrogen bonding between complementary subunits underpins their ability to encode and transmit information, to form sequence-defined duplexes, and to fold into catalytically active forms. Reversible covalent interactions between complementary subunits provide a different way to encode information, and potentially function, in sequence-defined oligomers. Here, we examine six oligoarylacetylene trimers composed of aniline and benzaldehyde subunits. Four of these trimers self-pair to form two-rung duplex structures, and two form macrocyclic 1,3-folded structures. The equilibrium proportions of these structures can be driven to favor each of the observed structures almost entirely depending upon the concentration of trimers and an acid catalyst. Quenching the acidic trimer solutions with an organic base kinetically traps all species such that they can be isolated and characterized. Mixtures of complementary trimers form exclusively sequence-specific 3-rung duplexes. Our results suggest that reversible covalent bonds could in principle guide the formation of more complex folded conformations of longer oligomers.

Hybridization and strand displacement kinetics determine the evolution of the base paired configurations of mixtures of oligonucleotides over time. Although much attention has been focused on the thermodynamics of DNA and RNA base pairing in the scientific literature, much less work has been done on the time dependence of interactions involving multiple strands, especially in RNA. Here we provide a study of oligoribonucleotide interaction kinetics and show that it is possible to calculate the association, dissociation and strand displacement rates displayed by short oligonucleotides (5nt-12nt) that exhibit no expected secondary structure as simple functions of oligonucleotide length, CG content, ΔG of hybridization and ΔG of toehold binding. We then show that the resultant calculated kinetic parameters are consistent with the experimentally observed time dependent changes in concentrations of the different species present in mixtures of multiple competing RNA strands. We show that by changing the mixture composition, it is possible to create and tune kinetic traps that extend by orders of magnitude the typical sub-second hybridization timescale of two complementary oligonucleotides. We suggest that the slow equilibration of complex oligonucleotide mixtures may have facilitated the nonenzymatic replication of RNA during the origin of life., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The first ribozymes are thought to have emerged at a time when RNA replication proceeded via nonenzymatic template copying processes. However, functional RNAs have stable folded structures, and such structures are much more difficult to copy than short unstructured RNAs. How can these conflicting requirements be reconciled? Also, how can the inhibition of ribozyme function by complementary template strands be avoided or minimized? Here, we show that short RNA duplexes with single-stranded overhangs can be converted into RNA stem loops by nonenzymatic cross-strand ligation. We then show that loop-closing ligation reactions enable the assembly of full-length functional ribozymes without any external template. Thus, one can envisage a potential pathway whereby structurally complex functional RNAs could have formed at an early stage of evolution when protocell genomes might have consisted only of collections of short replicating oligonucleotides.

Nonenzymatic template-directed RNA copying using chemically activated nucleotides is thought to have played a key role in the emergence of genetic information on the early Earth. A longstanding question concerns the number and nature of different environments that might have been necessary to enable all of the steps from nucleotide synthesis to RNA copying. Here we explore three sequential steps from this overall pathway: nucleotide activation, synthesis of imidazolium-bridged dinucleotides, and template-directed RNA copying. We find that all three steps can take place in one reaction mixture undergoing multiple freeze-thaw cycles. Recent experiments have demonstrated a potentially prebiotic methyl isocyanide-based nucleotide activation chemistry. However, the original version of this approach is incompatible with nonenzymatic RNA copying because the high required concentration of the imidazole activating group prevents the accumulation of the essential imidazolium-bridged dinucleotide. Here we report that ice eutectic phase conditions facilitate not only the methyl isocyanide-based activation of ribonucleotide 5′-monophosphates with stoichiometric 2-aminoimidazole, but also the subsequent conversion of these activated mononucleotides into imidazolium-bridged dinucleotides. Furthermore, this one-pot approach is compatible with template-directed RNA copying in the same reaction mixture. Our results suggest that the simple and common environmental fluctuation of freeze-thaw cycles could have played an important role in prebiotic nucleotide activation and nonenzymatic RNA copying.

Aminoacylated transfer RNAs, which harbor a covalent linkage between amino acids and RNA, are a universally conserved feature of life. Because they are essential substrates for ribosomal translation, aminoacylated oligonucleotides must have been present in the RNA world prior to the evolution of the ribosome. One possibility we are exploring is that the aminoacyl ester linkage served another function before being recruited for ribosomal protein synthesis. The nonenzymatic assembly of ribozymes from short RNA oligomers under realistic conditions remains a key challenge in demonstrating a plausible pathway from prebiotic chemistry to the RNA world. Here, we show that aminoacylated RNAs can undergo template-directed assembly into chimeric amino acid-RNA polymers that are active ribozymes. We demonstrate that such chimeric polymers can retain the enzymatic function of their all-RNA counterparts by generating chimeric hammerhead, RNA ligase, and aminoacyl transferase ribozymes. Amino acids with diverse side chains form linkages that are well tolerated within the RNA backbone and, in the case of an aminoacyl transferase, even in its catalytic center, potentially bringing novel functionalities to ribozyme catalysis. Our work suggests that aminoacylation chemistry may have played a role in primordial ribozyme assembly. Increasing the efficiency of this process provides an evolutionary rationale for the emergence of sequence and amino acid-specific aminoacyl-RNA synthetase ribozymes, which could then have generated the substrates for ribosomal protein synthesis., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

The identification of nonenzymatic pathways for nucleic acid replication is a key challenge in understanding the origin of life. We have previously shown that nonenzymatic RNA primer extension using 2-aminoimidazole (2AI) activated nucleotides occurs primarily through an imidazolium-bridged dinucleotide intermediate. The reactive nature and preorganized structure of the intermediate increase the efficiency of primer extension but remain insufficient to drive extensive copying of RNA templates containing all four canonical nucleotides. To understand the factors that limit RNA copying, we synthesized all ten 2AI-bridged dinucleotide intermediates and measured the kinetics of primer extension in a model system. The affinities of the ten dinucleotides for the primer/template/helper complexes vary by over 7,000-fold, consistent with nearest neighbor energetic predictions. Surprisingly, the reaction rates at saturating intermediate concentrations still vary by over 15-fold, with the most weakly binding dinucleotides exhibiting a lower maximal reaction rate. Certain noncanonical nucleotides can decrease sequence dependent differences in affinity and primer extension rate, while monomers bridged to short oligonucleotides exhibit enhanced binding and reaction rates. We suggest that more uniform binding and reactivity of imidazolium-bridged intermediates may lead to the ability to copy arbitrary template sequences under prebiotically plausible conditions., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The template-directed synthesis of RNA played an important role in the transition from prebiotic chemistry to the beginnings of RNA based life, but the mechanism of RNA copying chemistry is incompletely understood. We measured the kinetics of template copying with a set of primers with modified 3'-nucleotides and determined the crystal structures of these modified nucleotides in the context of a primer/template/substrate-analog complex. pH-rate profiles and solvent isotope effects show that deprotonation of the primer 3'-hydroxyl occurs prior to the rate limiting step, the attack of the alkoxide on the activated phosphate of the incoming nucleotide. The analogs with a 3 E ribose conformation show the fastest formation of 3'-5' phosphodiester bonds. Among those derivatives, the reaction rate is strongly correlated with the electronegativity of the 2'-substituent. We interpret our results in terms of differences in steric bulk and charge distribution in the ground vs. transition states., (© 2021 Wiley-VCH GmbH.)

Substitution of exocyclic oxygen with sulfur was shown to substantially influence the properties of RNA/DNA bases, which are crucial for prebiotic chemistry and photodynamic therapies. Upon UV irradiation, thionucleobases were shown to efficiently populate triplet excited states and can be involved in characteristic photochemistry or generation of singlet oxygen. Here, we show that the photochemistry of a thionucleobase can be considerably modified in a nucleoside, that is, by the presence of ribose. Our transient absorption spectroscopy experiments demonstrate that thiocytosine exhibits 5 times longer excited-state lifetime and different excited-state absorption features than thiocytidine. On the basis of accurate quantum chemical simulations, we assign these differences to the dominant population of a shorter-lived triplet nπ* state in the nucleoside and longer-lived triplet ππ* states in the nucleobase. This explains the distinctive photoanomerziation of thiocytidine and indicates that the nucleoside will be a less efficient phototherapeutic agent with regard to singlet oxygen generation.

Advances in origins of life research and prebiotic chemistry suggest that life as we know it may have emerged from an earlier RNA World. However, it has been difficult to reconcile the conditions used in laboratory experiments with real-world geochemical environments that may have existed on the early Earth and hosted the origin(s) of life. This challenge is due to geologic resurfacing and recycling that have erased the overwhelming majority of the Earth's prebiotic history. We therefore propose that Mars, a planet frozen in time, comprised of many surfaces that have remained relatively unchanged since their formation > 4 Gya, is the best alternative to search for environments consistent with geochemical requirements imposed by the RNA world. In this study, we synthesize in situ and orbital observations of Mars and modeling of its early atmosphere into solutions containing a range of pHs and concentrations of prebiotically relevant metals (Fe 2+ , Mg 2+ , and Mn 2+ ) spanning various candidate aqueous environments. We then experimentally determine RNA degradation kinetics due to metal-catalyzed hydrolysis (cleavage) and evaluate whether early Mars could have been permissive toward the accumulation of long-lived RNA polymers. Our results indicate that a Mg 2+ -rich basalt sourcing metals to a slightly acidic (pH 5.4) environment mediates the slowest rates of RNA cleavage, though geologic evidence and basalt weathering models suggest aquifers on Mars would be near neutral (pH ~ 7). Moreover, the early onset of oxidizing conditions on Mars has major consequences regarding the availability of oxygen-sensitive metals (i.e., Fe 2+ and Mn 2+ ) due to increased RNA degradation rates and precipitation. Overall, (a) low pH decreases RNA cleavage at high metal concentrations; (b) acidic to neutral pH environments with Fe 2+ or Mn 2+ cleave more RNA than Mg 2+ ; and (c) alkaline environments with Mg 2+ dramatically cleaves more RNA while precipitates were observed for Fe 2+ and Mn 2+ ., (© 2021 The Authors. Geobiology published by John Wiley & Sons Ltd.)

Nonenzymatic copying of RNA templates with activated nucleotides is a useful model for studying the emergence of heredity at the origin of life. Previous experiments with defined-sequence templates have pointed to the poor fidelity of primer extension as a major problem. Here we examine the origin of mismatches during primer extension on random templates in the simultaneous presence of all four 2-aminoimidazole-activated nucleotides. Using a deep sequencing approach that reports on millions of individual template-product pairs, we are able to examine correct and incorrect polymerization as a function of sequence context. We have previously shown that the predominant pathway for primer extension involves reaction with imidazolium-bridged dinucleotides, which form spontaneously by the reaction of two mononucleotides with each other. We now show that the sequences of correctly paired products reveal patterns that are expected from the bridged dinucleotide mechanism, whereas those associated with mismatches are consistent with direct reaction of the primer with activated mononucleotides. Increasing the ratio of bridged dinucleotides to activated mononucleotides, either by using purified components or by using isocyanide-based activation chemistry, reduces the error frequency. Our results point to testable strategies for the accurate nonenzymatic copying of arbitrary RNA sequences., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Recent advances in prebiotic chemistry are beginning to outline plausible pathways for the synthesis of the canonical ribonucleotides and their assembly into oligoribonucleotides. However, these reaction pathways suggest that many noncanonical nucleotides are likely to have been generated alongside the standard ribonucleotides. Thus, the oligomerization of prebiotically synthesized nucleotides is likely to have led to a highly heterogeneous collection of oligonucleotides comprised of a wide range of types of nucleotides connected by a variety of backbone linkages. How then did relatively homogeneous RNA emerge from this primordial heterogeneity? Here we focus on nonenzymatic template-directed primer extension as a process that would have strongly enriched for homogeneous RNA over the course of multiple cycles of replication. We review the effects on copying the kinetics of nucleotides with altered nucleobase and sugar moieties, when they are present as activated monomers and when they are incorporated into primer and template oligonucleotides. We also discuss three variations in backbone connectivity, all of which are nonheritable and regenerate native RNA upon being copied. The kinetic superiority of RNA synthesis suggests that nonenzymatic copying served as a chemical selection mechanism that allowed relatively homogeneous RNA to emerge from a complex mixture of prebiotically synthesized nucleotides and oligonucleotides.

UV light has been invoked as a source of energy for driving prebiotic chemistry, but such high energy photons are also known to cause damage to biomolecules and their precursors. One potential mechanism for increasing the lifetime of UV-photounstable molecules is to invoke a protection or shielding mechanism. UV shielding could either occur by the molecule in question itself (self-shielding) or by the presence of other UV-absorbing molecules. We investigate and illustrate these two shielding mechanisms as means of increasing the lifetime of 2-aminooxazole (AO), a prebiotic precursor molecule moderately susceptible to UV photodamage, with an expected half-life of 7 h on the surface of the early Earth. AO can be protected by being present in high concentrations, such that it self-shields. AO can similarly be protected by the presence of UV-absorbing nucleosides; the degree of protection depends on the concentration and identity of the nucleoside. The purine nucleosides (A, G, and I) confer more protection than the pyrimidines (C and U). We find that 0.1 mM purine ribonucleosides affords AO about the same protection as 1 mM AO self-shielding, corresponding to a lifetime enhancement of 2-3×. This suggests that only a modest yield of nucleosides can potentially allow for protection of UV photounstable molecules, and therefore this could be a plausible mechanism for protecting sensitive molecules while prebiotic synthesis is occurring simultaneously. Our findings suggest that both synthetic and degradative reactions can proceed at the same time, given various degrees of shielding., Competing Interests: The authors declare no competing financial interest., (© 2021 American Chemical Society.)

Aminoacylated tRNAs are the substrates for ribosomal protein synthesis in all branches of life, implying an ancient origin for aminoacylation chemistry. In the 1970s, Orgel and colleagues reported potentially prebiotic routes to aminoacylated nucleotides and their RNA-templated condensation to form amino acid-bridged dinucleotides. However, it is unclear whether such reactions would have aided or impeded non-enzymatic RNA replication. Determining whether aminoacylated RNAs could have been advantageous in evolution prior to the emergence of protein synthesis remains a key challenge. We therefore tested the ability of aminoacylated RNA to participate in both templated primer extension and ligation reactions. We find that at low magnesium concentrations that favor fatty acid-based protocells, these reactions proceed orders of magnitude more rapidly than when initiated from the cis -diol of unmodified RNA. We further demonstrate that amino acid-bridged RNAs can act as templates in a subsequent round of copying. Our results suggest that aminoacylation facilitated non-enzymatic RNA replication, thus outlining a potentially primordial functional link between aminoacylation chemistry and RNA replication.

The prebiotic synthesis of ribonucleotides is likely to have been accompanied by the synthesis of noncanonical nucleotides including the threo-nucleotide building blocks of TNA. Here, we examine the ability of activated threo-nucleotides to participate in nonenzymatic template-directed polymerization. We find that primer extension by multiple sequential threo-nucleotide monomers is strongly disfavored relative to ribo-nucleotides. Kinetic, NMR and crystallographic studies suggest that this is due in part to the slow formation of the imidazolium-bridged TNA dinucleotide intermediate in primer extension, and in part because of the greater distance between the attacking RNA primer 3'-hydroxyl and the phosphate of the incoming threo-nucleotide intermediate. Even a single activated threo-nucleotide in the presence of an activated downstream RNA oligonucleotide is added to the primer 10-fold more slowly than an activated ribonucleotide. In contrast, a single activated threo-nucleotide at the end of an RNA primer or in an RNA template results in only a modest decrease in the rate of primer extension, consistent with the minor and local structural distortions revealed by crystal structures. Our results are consistent with a model in which heterogeneous primordial oligonucleotides would, through cycles of replication, have given rise to increasingly homogeneous RNA strands., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

We propose a model for the replication of primordial protocell genomes that builds upon recent advances in the nonenzymatic copying of RNA. We suggest that the original genomes consisted of collections of oligonucleotides beginning and ending at all possible positions on both strands of one or more virtual circular sequences. Replication is driven by feeding with activated monomers and by the activation of monomers and oligonucleotides in situ. A fraction of the annealed configurations of the protocellular oligonucleotides would allow for template-directed oligonucleotide growth by primer extension or ligation. Rearrangements of these annealed configurations, driven either by environmental fluctuations or occurring spontaneously, would allow for continued oligonucleotide elongation. Assuming that shorter oligonucleotides were more abundant than longer ones, replication of the entire genome could occur by the growth of all oligonucleotides by as little as one nucleotide on average. We consider possible scenarios that could have given rise to such protocell genomes, as well as potential routes to the emergence of catalytically active ribozymes and thus the more complex cells of the RNA World., (© 2021 Zhou et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

The desire to create cell-like models for fundamental science and applications has spurred extensive effort toward creating giant unilamellar vesicles (GUVs). However, a route to selectively self-assemble GUVs in bulk has remained elusive. In bulk solution, membrane-forming molecules such as phospholipids, single-tailed surfactants, and block copolymers typically self-assemble into multilamellar, onion-like structures. So although self-assembly processes can form nanoscale unilamellar vesicles, scaffolding by droplets or surfaces is required to create GUVs. Here we show that it is possible to bulk self-assemble cell-sized GUVs with almost complete selectivity over other vesicle topologies. The seemingly paradoxical pair of features that enables this appears to be having very dynamic molecules at the nanoscale that create unusually rigid membranes. The resultant self-assembly pathway enables encapsulation of molecules and colloids and can also generate model primitive cells that can grow and divide.

Genetic biopolymers utilize defined sequences and monomer-specific molecular recognition to store and transfer information. Synthetic polymers that mimic these attributes using reversible covalent chemistry for base-pairing pose unique synthetic challenges. Here, we describe a solid-phase synthesis methodology for the efficient construction of ethynyl benzene oligomers with specific sequences of aniline and benzaldehyde subunits. Handling these oligomers is complicated by the fact that they often exhibit multiple conformations because of intra- or intermolecular pairing. We describe conditions that allow the dynamic behavior of these oligomers to be controlled so that they may be manipulated and characterized without needing to mask the recognition units with protecting groups.