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Quantum mechanical calculations are used to explore the thermodynamics of possible prebiotic synthesis of the building blocks of nucleic acids. Different combinations of D-ribofuranose (Ribf) and N-(2-aminoethyl)-glycine (AEG) (trifunctional connectors (TCs)); the nature of the Ribf, its anomeric form, and its ring puckering (conformation); and the nature of the nucleobases (recognition units (RUs)) are considered. The combinatorial explosion of possible nucleosides has been drastically reduced on physicochemical grounds followed by a detailed thermodynamic evaluation of alternative synthetic pathways. The synthesis of nucleosides containing N-(2-aminoethyl)-glycine (AEG) is predicted to be thermodynamically favored suggesting a possible role of AEG as a component of an ancestral proto-RNA that may have preceded today's nucleic acids. A new pathway for the building of free nucleotides (exemplified by 5'-uridine monophosphate (UMP)) and of AEG dipeptides is proposed. This new pathway leads to a spontaneous formation of free UMP assisted by an AEG nucleoside in an aqueous environment. This appears to be a workaround to the "water problem" that prohibits the synthesis of nucleotides in water., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

We hypothesize that the first ancestral "protocell" molecular structures, i.e., the first RNAs and peptides that gradually transformed into real cells once the Earth had cooled sufficiently for organic molecules to appear, have left traces in the RNAs and the genes in present cells. We propose a circular RNA that could have been one of these ancestral structures whose vestigial pentameric subsequences would mark the evolution from this key moment when the protocells began to join with living organisms. In particular, we propose that, in present RNAs (ribosomal or messenger), which play an important role in the metabolism of current cells, we look for traces of the proposed primitive structure in the form of pentamers (or longer fragments) that belong to their nucleotide sequence. The result obtained can be summarized in the existence of a gradient of occurrence of such pentamers, with a high frequency for the most vital functions (protein synthesis, nucleic synthesis, cell respiration, etc.). This gradient is also visible between organisms, from the oldest (Archaea) to the most recent (Eukaryotes) in the evolution of species.

Prebiotic pre-Darwinian reactions continued throughout biochemical or Darwinian evolution. Early chemical processes could have occurred on Earth between 4.5 and 3.6 billion years ago when cellular life was about to come into being. Pre-Darwinian evolution assumes the development of hereditary elements but does not regard them as self-organizing processes. The presence of biochemical self-organization after the pre-Darwinian evolution did not justify distinguishing between different types of evolution. From the many possible solutions, evolution selected from among those stable reactions that led to catalytic networks, and under gradually changing external conditions produced a reproducible, yet constantly evolving and adaptable, living system. Major abiotic factors included sunlight, precipitation, air, minerals, soil and the Earth's atmosphere, hydrosphere and lithosphere. Abiotic sources of chemicals contributed to the formation of prebiotic RNA, the development of genetic RNA, the RNA World and the initial life forms on Earth and the transition of genRNA to the DNA Empire, and eventually to the multitude of life forms today. The transition from the RNA World to the DNA Empire generated new processes such as oxygenic photosynthesis and the hierarchical arrangement of processes involved in the transfer of genetic information. The objective of this work is to unite earlier work dealing with the formose, the origin and synthesis of ribose and RNA reactions that were published as a series of independent reactions. These reactions are now regarded as the first metabolic pathway.

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.)

The obcell hypothesis is a proposed route for the RNA world to develop into a primitive cellular one. It posits that this transition began with the emergence of the proto-ribosome which enabled RNA to colonise the external surface of lipids by the synthesis of amphipathic peptidyl-RNAs. The obcell hypothesis also posits that the emergence of a predation-based ecosystem provided a selection mechanism for continued sophistication amongst early life forms. Here, I argue for this hypothesis owing to its significant explanatory power; it offers a rationale why a ribosome which initially was capable only of producing short non-coded peptides was advantageous and it forgoes issues related to maintaining a replicating RNA inside a lipid enclosure. I develop this model by proposing that the evolutionary selection for improved membrane anchors resulted in the emergence of primitive membrane pores which enabled obcells to gradually evolve into a cellular morphology. Moreover, I introduce a model of obcell production which advances that tRNAs developed from primers of the RNA world., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The world in which we live is homochiral. The ribose units that form the backbone of DNA and RNA are all D-configured and the encoded amino acids that comprise the proteins of all living species feature an all-L-configuration at the α-carbon atoms. The homochirality of α-amino acids is essential for folding of the peptides into well-defined and functional 3D structures and the homochirality of D-ribose is crucial for helix formation and base-pairing. The question of why nature uses only encoded L-α-amino acids is not understood. Herein, we show that an RNA-peptide world, in which peptides grow on RNAs constructed from D-ribose, leads to the self-selection of homo-L-peptides, which provides a possible explanation for the homo-D-ribose and homo-L-amino acid combination seen in nature., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Research on the origin of life investigates the transition from abiotic chemistry to the emergence of biology, with the 'RNA world hypothesis' as the leading theory. RNA's dual role in storage and catalysis suggests its importance in this narrative. The discovery of natural ribozymes emphasizes RNA's catalytic capabilities in prebiotic environments, supporting the plausibility of an RNA world and prompting exploration of precellular evolution. Collective autocatalytic sets (CASs) mark a crucial milestone in this transition, fostering complexity through autocatalysis. While modern biology emphasizes sequence-specific polymerases, remnants of CASs persist in primary metabolism highlighting their significance. Autocatalysis, driven by CASs, promotes complexity through mutually interdependent catalytic sets. Yet, the transition from ribonucleotides to complex RNA oligomers remains puzzling. Questions persist about the genesis of the first self-replicating RNA molecule, RNA's stability in prebiotic conditions, and the shift to complex molecular reproduction. This review delves into diverse facets of the RNA world's emergence, addressing critical bottlenecks and scientific advances. Integrating insights from simulation and in vitro evolution research, we illuminate the multistep biogenesis of catalytic RNA from the abiotic world. Through this exploration, we aim to elucidate the journey from the primordial soup to the dawn of life, emphasizing the interplay between chemistry and biology in understanding life's origins.

The RNA world hypothesis confers a central role to RNA molecules in information encoding and catalysis. Even though evidence in support of this hypothesis has accumulated from both experiments and computational modelling, the transition from an RNA world to a world where heritable genetic information is encoded in DNA remains an open question. Recent experiments show that both RNA and DNA templates can extend complementary primers using free RNA/DNA nucleotides, either non-enzymatically or in the presence of a replicase ribozyme. Guided by these experiments, we analyse protocellular evolution with an expanded set of reaction pathways made possible through the presence of DNA nucleotides. By encapsulating these reactions inside three different types of protocellular compartments, each subject to distinct modes of selection, we show how protocells containing DNA-encoded replicases in low copy numbers and replicases in high copy numbers can dominate the population. This is facilitated by a reaction that leads to auto-catalytic synthesis of replicase ribozymes from DNA templates encoding the replicase after the chance emergence of a replicase through non-enzymatic reactions. Our work unveils a pathway for the transition from an RNA world to a mixed RNA-DNA world characterized by Darwinian evolution, where DNA sequences encode heritable phenotypes.

It is likely that an RNA world existed in early life, when RNA played both the roles of the genome and functional molecules, thereby undergoing Darwinian evolution. However, even with only one type of polymer, it seems quite necessary to introduce a labour division concerning these two roles because folding is required for functional molecules (ribozymes) but unfavourable for the genome (as a template in replication). Notably, while ribozymes tend to have adopted a linear form for folding without constraints, a circular form, which might have been topologically hindered in folding, seems more suitable for an RNA template. Another advantage of involving a circular genome could have been to resist RNA's end-degradation. Here, we explore the scenario of a circular RNA genome plus linear ribozyme(s) at the precellular stage of the RNA world through computer modelling. The results suggest that a one-gene scene could have been 'maintained', albeit with rather a low efficiency for the circular genome to produce the ribozyme, which required precise chain-break or chain-synthesis. This strict requirement may have been relieved by introducing a 'noncoding' sequence into the genome, which had the potential to derive a second gene through mutation. A two-gene scene may have 'run well' with the two corresponding ribozymes promoting the replication of the circular genome from different respects. Circular genomes with more genes might have arisen later in RNA-based protocells. Therefore, circular genomes, which are common in the modern living world, may have had their 'root' at the very beginning of life.

Darwin's assertion that "it is mere rubbish thinking, at present, of origin of life" is no longer valid. By synthesizing origin of life (OoL) research from its inception to recent findings, with a focus on (i) proof-of-principle prebiotically plausible syntheses and (ii) molecular relics of the ancient RNA World, we present a comprehensive up-to-date description of science's understanding of the OoL and the RNA World hypothesis. Based on these observations, we solidify the consensus that RNA evolved before coded proteins and DNA genomes, such that the biosphere began with an RNA core where much of the translation apparatus and related RNA architecture arose before RNA transcription and DNA replication. This supports the conclusion that the OoL was a gradual process of chemical evolution involving a series of transitional forms between prebiotic chemistry and the last universal common ancestor (LUCA) during which RNA played a central role, and that many of the events and their relative order of occurrence along this pathway are known. The integrative nature of this synthesis also extends previous descriptions and concepts and should help inform future questions and experiments about the ancient RNA World and the OoL., (© 2023 Fine and Pearlman; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Trying to provide a broad overview about the origin of life in Earth, the most significant transitions of life before cells are listed and discussed. The current approach emphasizes the symbiotic relationships that emerged with life. We propose a rational, stepwise scenario for the origin of life that starts with the origin of the first biomolecules and steps forward until the origins of the first cells. Along this path, we aim to provide a brief, though comprehensive theoretical model that will consider the following steps: (i) how nucleotides and other biomolecules could be made prebiotically in specific prebiotic refuges; (ii) how the first molecules of RNAs were formed; (iii) how the proto-peptidyl transferase center was built by the concatenation of proto-tRNAs; (iv) how the ribosome and the genetic code could be structured; (v) how progenotes could live and reproduce as "naked" ribonucleoprotein molecules; (vi) how peptides started to bind molecules in the prebiotic soup allowing biochemical pathways to evolve from those bindings; (vii) how genomes got bigger by the symbiotic relationship of progenotes and lateral transference of genetic material; (viii) how the progenote LUCA has been formed by assembling most biochemical routes; (ix) how the first virion capsids probably emerged and evolved; (x) how phospholipid membranes emerged probably twice by the evolution of lipid-binding proteins; (xi) how DNA synthesis have been formed in parallel in Bacteria and Archaea; and, finally, (xii) how DNA-based cells of Bacteria and Archaea have been constituted. The picture provided is conjectural and present epistemological gaps. Future research will help to advance into the elucidation of gaps and confirmation/refutation of current statements., Competing Interests: Declaration of competing interest We declare that none people or organizations inappropriately influenced our work., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

RNA is a molecule that can both store genetic information and perform catalytic reactions. This observed dualism places RNA into the limelight of concepts about the origin of life. The RNA world concept argues that life started from self-replicating RNA molecules, which evolved toward increasingly complex structures. Recently, we demonstrated that RNA, with the help of conserved non-canonical nucleosides, which are also putative relics of an early RNA world, had the ability to grow peptides covalently connected to RNA nucleobases, creating RNA-peptide chimeras. It is conceivable that such molecules, which combined the information-coding properties of RNA with the catalytic potential of amino acid side chains, were once the structures from which life emerged. Herein, we report prebiotic chemistry that enabled the loading of both nucleosides and RNAs with amino acids as the first step toward RNA-based peptide synthesis in a putative RNA-peptide world., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

How life emerged from simple non-life chemicals on the ancient Earth is one of the greatest mysteries in biology. The gene expression system of extant life is based on the interdependence between multiple molecular species (DNA, RNA, and proteins). While DNA is mainly used as genetic material and proteins as functional molecules in modern biology, RNA serves as both genetic material and enzymes (ribozymes). Thus, the evolution of life may have begun with the birth of a ribozyme that replicated itself (the RNA world hypothesis), and proteins and DNA joined later. However, the complete self-replication of ribozymes from monomeric substrates has not yet been demonstrated experimentally, due to their limited activity and stability. In contrast, peptides are more chemically stable and are considered to have existed on the ancient Earth, leading to the hypothesis of RNA-peptide co-evolution from the very beginning. Our group and collaborators recently demonstrated that (1) peptides with both hydrophobic and cationic moieties (e.g., KKVVVVVV) form β-amyloid aggregates that adsorb RNA and enhance RNA synthesis by an artificial RNA polymerase ribozyme and (2) a simple peptide with only seven amino acid types (especially rich in valine and lysine) can fold into the ancient β-barrel conserved in various enzymes, including the core of cellular RNA polymerases. These findings, together with recent reports from other groups, suggest that simple prebiotic peptides could have supported the ancient RNA-based replication system, gradually folded into RNA-binding proteins, and eventually evolved into complex proteins like RNA polymerase., (© 2023 Japanese Society of Developmental Biologists.)

The modern ribosome catalyzes all coded protein synthesis in extant organisms. It is likely that its core structure is a direct descendant from the ribosome present in the last common ancestor (LCA). Hence, its earliest origins likely predate the LCA and therefore date further back in time. Of special interest is the pseudosymmetrical region (SymR) that lies deep within the large subunit (LSU) where the peptidyl transfer reaction takes place. It was previously proposed that two RNA oligomers, representing the P- and A-regions of extant ribosomes dimerized to create a pore-like structure, which hosted the necessary properties that facilitate peptide bond formation. However, recent experimental studies show that this may not be the case. Instead, several RNA constructs derived exclusively from the P-region were shown to form a homodimer capable of peptide bond synthesis. Of special interest will be the origin issues because the homodimer would have allowed a pre-LCA ribosome that was significantly smaller than previously proposed. For the A-region, the immediate issue will likely be its origin and whether it enhances ribosome performance. Here, we reanalyze the RNA/RNA interaction regions that most likely lead to SymR formation in light of these recent findings. Further, it has been suggested that the ability of these RNA constructs to dimerize and enhance peptide bond formation is sequence-dependent. We have analyzed the implications of sequence variations as parts of functional and nonfunctional constructs., (© 2023 Rivas and Fox; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

There are several theories on the origin of life, which differ by choosing the preponderant factor of emergence: main function (autocatalysis versus replication), initial location (black smokers versus ponds) or first molecule (RNA versus DNA). Among the two last ones, the first assumes that an RNA world involving a collaboration of small RNAs with amino-acids pre-existed and the second that DNA-enzyme-lipid complexes existed first. The debate between these classic theories is not closed and the arguments for one or the other of these theories have recently fueled a debate in which the two have a high degree of likelihood. It therefore seems interesting to propose a third intermediate way, based on the existence of an RNA that may have existed before the latter stages postulated by these theories, and therefore may be the missing link towards a common origin of them. To search for a possible ancestral structure, we propose as candidate a small RNA existing in ring or hairpin form in the early stages of life, which could have acted as a "proto-ribosome" by favoring the synthesis of the first peptides. Remnants of this putative candidate RNA exist in molecules nowadays involved in the ribosomal factory, the concentrations of these relics depending on the seniority of these molecules within the translation process.

When water interacts with porous rocks, its wetting and surface tension properties create air bubbles in large number. To probe their relevance as a setting for the emergence of life, we microfluidically created foams that were stabilized with lipids. A persistent non-equilibrium setting was provided by a thermal gradient. The foam's large surface area triggers capillary flows and wet-dry reactions that accumulate, aggregate and oligomerize RNA, offering a compelling habitat for RNA-based early life as it offers both wet and dry conditions in direct neighborhood. Lipids were screened to stabilize the foams. The prebiotically more probable myristic acid stabilized foams over many hours. The capillary flow created by the evaporation at the water-air interface provided an attractive force for molecule localization and selection for molecule size. For example, self-binding oligonucleotide sequences accumulated and formed micrometer-sized aggregates which were shuttled between gas bubbles. The wet-dry cycles at the foam bubble interfaces triggered a non-enzymatic RNA oligomerization from 2',3'-cyclic CMP and GMP which despite the small dry reaction volume was superior to the corresponding dry reaction. The found characteristics make heated foams an interesting, localized setting for early molecular evolution., (© 2022 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

The question of how RNA, as the principal carrier of genetic information evolved is fundamentally important for our understanding of the origin of life. The RNA molecule is far too complex to have formed in one evolutionary step, suggesting that ancestral proto-RNAs (first ancestor of RNA) may have existed, which evolved over time into the RNA of today. Here we show that isoxazole nucleosides, which are quickly formed from hydroxylamine, cyanoacetylene, urea and ribose, are plausible precursors for RNA. The isoxazole nucleoside can rearrange within an RNA-strand to give cytidine, which leads to an increase of pairing stability. If the proto-RNA contains a canonical seed-nucleoside with defined stereochemistry, the seed-nucleoside can control the configuration of the anomeric center that forms during the in-RNA transformation. The results demonstrate that RNA could have emerged from evolutionarily primitive precursor isoxazole ribosides after strand formation., (© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

The synthesis of complementary strands is the reaction underlying the replication of genetic information. It is likely that the earliest self-replicating systems used RNA as genetic material. How RNA was copied in the absence of enzymes and what sequences were most likely to have supported replication is not clear. Here we show that mixtures of dinucleotides with C and G as bases copy an RNA sequence of up to 12 nucleotides in dilute aqueous solution. Successful enzyme-free copying occurred with in situ activation at 4 °C and pH 6.0. Dimers were incorporated in favor of monomers when both competed as reactants, and little misincorporation was detectable in mass spectra. Simulations using experimental rate constants confirmed that mixed C/G sequences are good candidates for successful replication with dimers. Because dimers are intermediates in the synthesis of longer strands, our results support evolutionary scenarios encompassing formation and copying of RNA strands in enzyme-free fashion., (© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Reported here are experiments that show that ribonucleoside triphosphates are converted to polyribonucleic acid when incubated with rock glasses similar to those likely present 4.3-4.4 billion years ago on the Hadean Earth surface, where they were formed by impacts and volcanism. This polyribonucleic acid averages 100-300 nucleotides in length, with a substantial fraction of 3',-5'-dinucleotide linkages. Chemical analyses, including classical methods that were used to prove the structure of natural RNA, establish a polyribonucleic acid structure for these products. The polyribonucleic acid accumulated and was stable for months, with a synthesis rate of 2 × 10 -3 pmoles of triphosphate polymerized each hour per gram of glass (25°C, pH 7.5). These results suggest that polyribonucleotides were available to Hadean environments if triphosphates were. As many proposals are emerging describing how triphosphates might have been made on the Hadean Earth, the process observed here offers an important missing step in models for the prebiotic synthesis of RNA.

The RNA world concept 1 is one of the most fundamental pillars of the origin of life theory 2-4 . It predicts that life evolved from increasingly complex self-replicating RNA molecules 1,2,4 . The question of how this RNA world then advanced to the next stage, in which proteins became the catalysts of life and RNA reduced its function predominantly to information storage, is one of the most mysterious chicken-and-egg conundrums in evolution 3-5 . Here we show that non-canonical RNA bases, which are found today in transfer and ribosomal RNAs 6,7 , and which are considered to be relics of the RNA world 8-12 , are able to establish peptide synthesis directly on RNA. The discovered chemistry creates complex peptide-decorated RNA chimeric molecules, which suggests the early existence of an RNA-peptide world 13 from which ribosomal peptide synthesis 14 may have emerged 15,16 . The ability to grow peptides on RNA with the help of non-canonical vestige nucleosides offers the possibility of an early co-evolution of covalently connected RNAs and peptides 13,17,18 , which then could have dissociated at a higher level of sophistication to create the dualistic nucleic acid-protein world that is the hallmark of all life on Earth., (© 2022. The Author(s).)

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.

In prebiotic evolution, self-replicating molecules are believed to have evolved into complex living systems by expanding their information and functions open-endedly. Theoretically, such evolutionary complexification could occur through successive appearance of novel replicators that interact with one another to form replication networks. Here we perform long-term evolution experiments of RNA that replicates using a self-encoded RNA replicase. The RNA diversifies into multiple coexisting host and parasite lineages, whose frequencies in the population initially fluctuate and gradually stabilize. The final population, comprising five RNA lineages, forms a replicator network with diverse interactions, including cooperation to help the replication of all other members. These results support the capability of molecular replicators to spontaneously develop complexity through Darwinian evolution, a critical step for the emergence of life., (© 2022. The Author(s).)

Although the mode of action of the ribosomes, the multi-component universal effective protein-synthesis organelles, has been thoroughly explored, their mere appearance remained elusive. Our earlier comparative structural studies suggested that a universal internal small RNA pocket-like segment called by us the protoribosome, which is still embedded in the contemporary ribosome, is a vestige of the primordial ribosome. Herein, after constructing such pockets, we show using the "fragment reaction" and its analyses by MALDI-TOF and LC-MS mass spectrometry techniques, that several protoribosome constructs are indeed capable of mediating peptide-bond formation. These findings present strong evidence supporting our hypothesis on origin of life and on ribosome's construction, thus suggesting that the protoribosome may be the missing link between the RNA dominated world and the contemporary nucleic acids/proteins life., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

RNA-catalyzed RNA replication is widely considered a key step in the emergence of life's first genetic system. However, RNA replication can be impeded by the extraordinary stability of duplex RNA products, which must be dissociated for re-initiation of the next replication cycle. Here, we have explored rolling circle synthesis (RCS) as a potential solution to this strand separation problem. We observe sustained RCS by a triplet polymerase ribozyme beyond full-length circle synthesis with strand displacement yielding concatemeric RNA products. Furthermore, we show RCS of a circular Hammerhead ribozyme capable of self-cleavage and re-circularization. Thus, all steps of a viroid-like RNA replication pathway can be catalyzed by RNA alone. Finally, we explore potential RCS mechanisms by molecular dynamics simulations, which indicate a progressive build-up of conformational strain upon RCS with destabilization of nascent strand 5'- and 3'-ends. Our results have implications for the emergence of RNA replication and for understanding the potential of RNA to support complex genetic processes., Competing Interests: EK, MB, AN, PH No competing interests declared, (© 2022, Kristoffersen et al.)

This review covers research findings reported over the past decade concerning the ability of low complexity (LC) domains to self-associate in a manner leading to their phase separation from aqueous solution. We focus our message upon the reductionist use of two forms of phase separation as biochemical assays to study how LC domains might function in living cells. Cells and their varied compartments represent extreme examples of material condensates. Over the past half century, biochemists, structural biologists, and molecular biologists have resolved the mechanisms driving innumerable forms of macromolecular condensation. In contrast, we remain largely ignorant as to how 10%-20% of our proteins actually work to assist in cell organization. This enigmatic 10%-20% of the proteome corresponds to gibberish-like LC sequences. We contend that many of these LC sequences move in and out of a structurally ordered, self-associated state as a means of offering a combination of organizational specificity and dynamic pliability to living cells. Finally, we speculate that ancient proteins may have behaved similarly, helping to condense, organize, and protect RNA early during evolution., (© 2022 Kato et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

The hypothesis that prebiotic molecules were transformed into polymers that evolved into proliferating molecular assemblages and eventually a primitive cell was first proposed about 100 years ago. To the best of our knowledge, however, no model of a proliferating prebiotic system has yet been realised because different conditions are required for polymer generation and self-assembly. In this study, we identify conditions suitable for concurrent peptide generation and self-assembly, and we show how a proliferating peptide-based droplet could be created by using synthesised amino acid thioesters as prebiotic monomers. Oligopeptides generated from the monomers spontaneously formed droplets through liquid-liquid phase separation in water. The droplets underwent a steady growth-division cycle by periodic addition of monomers through autocatalytic self-reproduction. Heterogeneous enrichment of RNA and lipids within droplets enabled RNA to protect the droplet from dissolution by lipids. These results provide experimental constructs for origins-of-life research and open up directions in the development of peptide-based materials., (© 2021. The Author(s).)

The RNA world hypothesis relies on the double-helix complementarity principle for both replication and catalytic activity of RNA. However, the de novo appearance of the complementarity rules, without previous evolution steps, is doubtful. Another major problem of the RNA world is its isolated nature, making it almost impossible to accommodate the genetic code and transform it into modern biochemistry. These and many other unanswered questions of the RNA world led to suggestions that some simpler molecules must have preceded RNA. Most of these alternative hypotheses proposed the double-helical polymers with different backbones but used the same complementarity principle. The current paper describes a fundamentally different idea: the de novo appearance of a nucleic acid polymer without any preexisting rules or requirements. This approach, coined as the quadruplex world hypothesis, is based on (i) the ability of guanines to form stable G-tetrads that facilitate polymerization; and (ii) the unique property of polyguanines to fold into a monomolecular tetrahelix with a strictly defined building pattern and tertiary structure. The tetrahelix is capable of high-affinity intermolecular interactions and catalytic activities. The quadruplex world hypothesis has the potential to address almost all the shortcomings of the RNA world., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Structural relations in an evolutionary context of polymerases is crucial to gain insights into the transition from an RNA world to a Ribonucleoprotein world. Herein, we present a structural proximity tree for the polymerases, from which we observe that the enzymes that have RNA as substrate are more homogeneous than the group with DNA as substrate. The homogeneity observed in enzymes with RNA as a substrate, may be because they performed all steps in information processing. In this sense, the emergence of the DNA molecule posed new challenges to the biological systems, where several parts of the informational flow were individualized by the emergence of enzymes for each step. From the data presented, we propose a polymerase diversification model, in which we have RNA-dependent RNA polymerases as an ancestor and all other polymerases diverged directly from this group by a radiation process., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The synthetic pathways of life's building blocks are envisaged to be through a series of complex prebiotic reactions and processes. However, the strategy to compartmentalize and concentrate biopolymers under prebiotic conditions remains elusive. Liquid-liquid phase separation is a mechanism by which membraneless organelles form inside cells, and has been hypothesized as a potential mechanism for prebiotic compartmentalization. Associative phase separation of oppositely charged species has been shown to partition RNA, but the strongly negative charge exhibited by RNA suggests that RNA-polycation interactions could inhibit RNA folding and its functioning inside the coacervates. Here, we present a prebiotically plausible pathway for non-associative phase separation within an evaporating all-aqueous sessile droplet. We quantitatively investigate the kinetic pathway of phase separation triggered by the non-uniform evaporation rate, together with the Marangoni flow-driven hydrodynamics inside the sessile droplet. With the ability to undergo liquid-liquid phase separation, the drying droplets provide a robust mechanism for formation of prebiotic membraneless compartments, as demonstrated by localization and storage of nucleic acids, in vitro transcription, as well as a three-fold enhancement of ribozyme activity. The compartmentalization mechanism illustrated in this model system is feasible on wet organophilic silica-rich surfaces during early molecular evolution.

The theory of chemical symbiosis (TCS) suggests that biological systems started with the collaboration of two polymeric molecules existing in early Earth: nucleic acids and peptides. Chemical symbiosis emerged when RNA-like nucleic acid polymers happened to fold into 3D structures capable to bind amino acids together, forming a proto peptidyl-transferase center. This folding catalyzed the formation of quasi-random small peptides, some of them capable to bind this ribozyme structure back and starting to form an initial layer that would produce the larger subunit of the ribosome by accretion. TCS suggests that there is no chicken-and-egg problem into the emergence of biological systems as RNAs and peptides were of equal importance to the origin of life. Life has initially emerged when these two macromolecules started to interact in molecular symbiosis. Further, we suggest that life evolved into progenotes and cells due to the emergence of new layers of symbiosis. Mutualism is the strongest force in biology, capable to create novelties by emergent principles; on which the whole is bigger than the sum of the parts. TCS aims to apply the Margulian view of biology into the origins of life field.

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.)

Urea appears to be a key intermediate of important prebiotic synthetic pathways. Concentrated pools of urea likely existed on the surface of the early Earth, as urea is synthesized in significant quantities from hydrogen cyanide or cyanamide (widely accepted prebiotic molecules), it has extremely high water solubility, and it can concentrate to form eutectics from aqueous solutions. We propose a model for the origin of a variety of canonical and non-canonical nucleobases, including some known to form supramolecular assemblies that contain Watson-Crick-like base pairs.The dual nucleophilic-electrophilic character of urea makes it an ideal precursor for the formation of nitrogenous heterocycles. We propose a model for the origin of a variety of canonical and noncanonical nucleobases, including some known to form supramolecular assemblies that contain Watson-Crick-like base pairs. These reactions involve urea condensation with other prebiotic molecules (e. g., malonic acid) that could be driven by environmental cycles (e. g., freezing/thawing, drying/wetting). The resulting heterocycle assemblies are compatible with the formation of nucleosides and, possibly, the chemical evolution of molecular precursors to RNA. We show that urea eutectics at moderate temperature represent a robust prebiotic source of nitrogenous heterocycles. The simplicity of these pathways, and their independence from specific or rare geological events, support the idea of urea being of fundamental importance to the prebiotic chemistry that gave rise to life on Earth., (© 2020 Wiley-VCH GmbH.)

Fossils of extinct species allow us to reconstruct the process of Darwinian evolution that led to the species diversity we see on Earth today. The origin of the first functional molecules able to undergo molecular evolution and thus eventually able to create life, are largely unknown. The most prominent idea in the field posits that biology was preceded by an era of molecular evolution, in which RNA molecules encoded information and catalysed their own replication. This RNA world concept stands against other hypotheses, that argue for example that life may have begun with catalytic peptides and primitive metabolic cycles. The question whether RNA or peptides were first is addressed by the RNA-peptide world concept, which postulates a parallel existence of both molecular species. A plausible experimental model of how such an RNA-peptide world may have looked like, however, is absent. Here we report the synthesis and physicochemical evaluation of amino acid containing adenosine bases, which are closely related to molecules that are found today in the anticodon stem-loop of tRNAs from all three kingdoms of life. We show that these adenosines lose their base pairing properties, which allow them to equip RNA with amino acids independent of the sequence context. As such we may consider them to be living molecular fossils of an extinct molecular RNA-peptide world., (© 2020 The Authors. Published by Wiley-VCH GmbH.)

Life is an out-of-equilibrium system sustained by a continuous supply of energy. In extant biology, the generation of the primary energy currency, adenosine 5'-triphosphate and its use in the synthesis of biomolecules require enzymes. Before their emergence, alternative energy sources, perhaps assisted by simple catalysts, must have mediated the activation of carboxylates and phosphates for condensation reactions. Here, we show that the chemical energy inherent to isonitriles can be harnessed to activate nucleoside phosphates and carboxylic acids through catalysis by acid and 4,5-dicyanoimidazole under mild aqueous conditions. Simultaneous activation of carboxylates and phosphates provides multiple pathways for the generation of reactive intermediates, including mixed carboxylic acid-phosphoric acid anhydrides, for the synthesis of peptidyl-RNAs, peptides, RNA oligomers and primordial phospholipids. Our results indicate that unified prebiotic activation chemistry could have enabled the joining of building blocks in aqueous solution from a common pool and enabled the progression of a system towards higher complexity, foreshadowing today's encapsulated peptide-nucleic acid system.

Over 200 molecules have been detected in multiple extraterrestrial environments, including glycolaldehyde (C 2 (H 2 O) 2 , GLA), a two-carbon sugar precursor that has been detected in regions of the interstellar medium. Its recent in situ detection on the nucleus of comet 67P/Churyumov-Gerasimenko and through remote observations in the comae of others provides tantalizing evidence that it is common on most (if not all) comets. Impact experiments conducted at the Experimental Impact Laboratory at NASA's Johnson Space Center have shown that samples of GLA and GLA mixed with montmorillonite clays can survive impact delivery in the pressure range of 4.5 to 25 GPa. Extrapolated to amounts of GLA observed on individual comets and assuming a monotonic impact rate in the first billion years of Solar System history, these experimental results show that up to 10 23 kg of cometary GLA could have survived impact delivery, with substantial amounts of threose, erythrose, glycolic acid, and ethylene glycol also produced or delivered. Importantly, independent of the profile of the impact flux in the early Solar System, comet delivery of GLA would have provided (and may continue to provide) a reservoir of starting material for the formose reaction (to form ribose) and the Strecker reaction (to form amino acids). Thus, comets may have been important delivery vehicles for starting molecules necessary for life as we know it.

A major challenge in understanding how biological cells arose on the early Earth is explaining how RNA and membranes originally colocalized. We propose that the building blocks of RNA (nucleobases and ribose) bound to self-assembled prebiotic membranes. We have previously demonstrated that the bases bind to membranes composed of a prebiotic fatty acid, but evidence for the binding of sugars has remained a technical challenge. Here, we used pulsed-field gradient NMR spectroscopy to demonstrate that ribose and other sugars bind to membranes of decanoic acid. Moreover, the binding of some bases is strongly enhanced when they are linked to ribose to form a nucleoside or - with the addition of phosphate - a nucleotide. This enhanced binding could have played a role in the molecular evolution leading to the production of RNA., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In the past decade, astrochemistry has witnessed an impressive increase in the number of detections of complex organic molecules. Some of these species are of prebiotic interest such as glycolaldehyde, the simplest sugar, or aminoacetonitrile, a possible precursor of glycine. Recently, we have reported the detection of two new nitrogen-bearing complex organics, glycolonitrile and Z-cyanomethanimine, known to be intermediate species in the formation process of ribonucleotides within theories of a primordial RNA-world for the origin of life. In this study, we present deep and high-sensitivity observations toward two of the most chemically rich sources in the galaxy: a giant molecular cloud in the center of the Milky Way (G + 0.693-0.027) and a proto-Sun (IRAS16293-2422 B) . Our aim is to explore whether the key precursors considered to drive the primordial RNA-world chemistry are also found in space. Our high-sensitivity observations reveal that urea is present in G + 0.693-0.027 with an abundance of ∼5 × 10 -11 . This is the first detection of this prebiotic species outside a star-forming region. Urea remains undetected toward the proto-Sun IRAS16293-2422 B (upper limit to its abundance of ≤2 × 10 -11 ). Other precursors of the RNA-world chemical scheme such as glycolaldehyde or cyanamide are abundant in space, but key prebiotic species such as 2-amino-oxazole, glyceraldehyde, or dihydroxyacetone are not detected in either source. Future more sensitive observations targeting the brightest transitions of these species will be needed to disentangle whether these large prebiotic organics are certainly present in space.

The origins of life on Earth have been the subject of inquiry since the early days of philosophical thought and are still intensively investigated by the researchers around the world. One of the theories explaining the life emergence, that gained the most attention recently is the RNA World hypothesis, which assumes that life on Earth was sparked by replicating RNA chains. Since wet lab analysis is time-consuming, many mathematical and computational approaches have been proposed that try to explain the origins of life. Recently proposed one, based on the work by Takeuchi and Hogeweg, addresses the problem of interplay between RNA replicases and RNA parasitic species, which is crucial for understanding the first steps of prebiotic evolution. In this paper, the aforementioned model has been extended and modified by introducing RNA sequence (structure) information and mutation rate close to real one. It allowed to observe the simple evolution mechanisms, which could have led to the more complicated systems and eventually, to the formation of the first cells. The main goal of this study was to determine the conditions that allowed the spontaneous emergence and evolution of the prebiotic replicases equipped with simple functional domains within a large population. Here we show that polymerase ribozymes could have appeared randomly and then quickly started to copy themselves in order for the system to reach equilibrium. It has been shown that evolutionary selection works even in the simplest systems., Competing Interests: The authors have declared that no competing interests exist.

The close synergy between peptides and nucleic acids in current biology is suggestive of a functional co-evolution between the two polymers. Here we show that cationic proto-peptides (depsipeptides and polyesters), either produced as mixtures from plausibly prebiotic dry-down reactions or synthetically prepared in pure form, can engage in direct interactions with RNA resulting in mutual stabilization. Cationic proto-peptides significantly increase the thermal stability of folded RNA structures. In turn, RNA increases the lifetime of a depsipeptide by >30-fold. Proto-peptides containing the proteinaceous amino acids Lys, Arg, or His adjacent to backbone ester bonds generally promote RNA duplex thermal stability to a greater magnitude than do analogous sequences containing non-proteinaceous residues. Our findings support a model in which tightly-intertwined biological dependencies of RNA and protein reflect a long co-evolutionary history that began with rudimentary, mutually-stabilizing interactions at early stages of polypeptide and nucleic acid co-existence.

Continuous reaction networks, which do not rely on purification or timely additions of reagents, serve as models for chemical evolution and have been demonstrated for compounds thought to have played important roles for the origins of life such as amino acids, hydroxy acids, and sugars. Step-by-step chemical protocols for ribonucleotide synthesis are known, but demonstrating their synthesis in the context of continuous reaction networks remains a major challenge. Herein, compounds proposed to be important for prebiotic RNA synthesis, including glycolaldehyde, cyanamide, 2-aminooxazole, and 2-aminoimidazole, are generated from a continuous reaction network, starting from an aqueous mixture of NaCl, NH 4 Cl, phosphate, and HCN as the only carbon source. No well-timed addition of any other reagents is required. The reaction network is driven by a combination of γ radiolysis and dry-down. γ Radiolysis results in a complex mixture of organics, including the glycolaldehyde-derived glyceronitrile and cyanamide. This mixture is then dried down, generating free glycolaldehyde that then reacts with cyanamide/NH 3 to furnish a combination of 2-aminooxazole and 2-aminoimidazole. This continuous reaction network models how precursors for generating RNA and other classes of compounds may arise spontaneously from a complex mixture that originates from simple reagents., Competing Interests: The authors declare no competing interest.

The nature of the first genetic polymer is the subject of major debate 1 . Although the 'RNA world' theory suggests that RNA was the first replicable information carrier of the prebiotic era-that is, prior to the dawn of life 2,3 -other evidence implies that life may have started with a heterogeneous nucleic acid genetic system that included both RNA and DNA 4 . Such a theory streamlines the eventual 'genetic takeover' of homogeneous DNA from RNA as the principal information-storage molecule, but requires a selective abiotic synthesis of both RNA and DNA building blocks in the same local primordial geochemical scenario. Here we demonstrate a high-yielding, completely stereo-, regio- and furanosyl-selective prebiotic synthesis of the purine deoxyribonucleosides: deoxyadenosine and deoxyinosine. Our synthesis uses key intermediates in the prebiotic synthesis of the canonical pyrimidine ribonucleosides (cytidine and uridine), and we show that, once generated, the pyrimidines persist throughout the synthesis of the purine deoxyribonucleosides, leading to a mixture of deoxyadenosine, deoxyinosine, cytidine and uridine. These results support the notion that purine deoxyribonucleosides and pyrimidine ribonucleosides may have coexisted before the emergence of life 5 .

Evolution requires self-replication. But, what was the very first self-replicator directly ancestral to all life? The currently favoured RNA World theory assigns this role to RNA alone but suffers from a number of seemingly intractable problems. Instead, we suggest that the self-replicator consisted of both peptides and nucleic acid strands. Such a nucleopeptide replicator is more feasible both in the light of the replication machinery currently found in cells and the complexity of the evolutionary path required to reach them. Recent theoretical and mathematical work supports this idea and provide a blueprint for future investigations., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Despite decades of intensive research, many questions remain on the formation and growth of the first cells on Earth. Here, we used computer simulation to compare the self-assembly process of ribonucleic acids in two environments: enclosed in a vesicle-cell membrane and in the bulk. The self-assembly was found to be more favoured in the former environment, and the origin of such a biointerface effect was identified. These results will contribute to a better understanding of the origin of life on the primitive Earth.

The chemical or prebiotic evolution referred also to as pre-Darwinian evolution describes chemical reactions up to the origin of a self-replicating system that was capable of Darwinian evolution. These chemical processes took place on Earth between about 3.7 and 4.5 billion years ago when cellular life came into being. The pre-Darwinian chemical evolution usually assumes hereditary elements, but does not regard them as self-organizing processes. Physical and chemical self-organization led to uninterrupted pre-Darwinian and Darwinian evolution. Thus, it is not justified to distinguish between different types of evolution. From the many possible solutions, evolution selected among those reactions that generated catalytic networks incorporating chemical sequence information and under gradually changing circumstances produced a reproducible and stable living system that adapted to these conditions. Major issues in this review involve prebiotic reactions leading to genetic evolution involving (1) abiotic sources of components of ribonucleotides and xenobiotic nucleotides, (2) formation of prebiotic RNA, (3) development of genetic RNA from random-sequence noncoding RNA, (4) transition from RNA World to DNA Empire, (5) the role of oxygenic photosynthesis in genetic transitions, and (6) hierarchical arrangement of processes involved in the optimized genetic system.

Most scenarios for the origin of life assume that RNA played a key role in both catalysis and information storage. The A, U, G, and C nucleobases in modern RNA all participate in secondary structure formation and replication. However, the rapid deamination of C to U and the absence of C in meteorite samples suggest that prebiotic RNA may have been deficient in cytosine. Here, we assess the ability of RNA sequences formed from a three-letter AUG alphabet to perform both structural and genetic roles in comparison to sequences formed from the AUGC alphabet. Despite forming less thermodynamically stable helices, the AUG alphabet can find a broad range of structures and thus appears sufficient for catalysis in the RNA World. However, in the AUG case, longer sequences are required to form structures with an equivalent complexity. Replication in the AUG alphabet requires GU pairing. Sequence fidelity in the AUG alphabet is low whenever G's are present in the sequence. We find that AUG sequences evolve to AU sequences if GU pairing is rare, and to RU sequences if GU pairing is common (R denotes A or G). It is not possible to conserve a G at a specific site in either case. These problems do not rule out the possibility of an RNA World based on AUG, but they show that it wouldbe significantly more difficult than with a four-base alphabet., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The RNA world hypothesis assumes that life on Earth began with nucleotides that formed information-carrying RNA oligomers able to self-replicate. Prebiotic reactions leading to the contemporary nucleosides are now known, but their execution often requires specific starting materials and lengthy reaction sequences. It was therefore proposed that the RNA world was likely proceeded by a proto-RNA world constructed from molecules that were likely present on the early Earth in greater abundance. Herein, we show that the prebiotic starting molecules bis-urea (biuret) and tris-urea (triuret) are able to directly react with ribose. The urea-ribosides are remarkably stable because they are held together by a network of intramolecular, bifurcated hydrogen bonds. This even allowed the synthesis of phosphoramidite building blocks and incorporation of the units into RNA. Investigations of the nucleotides' base-pairing potential showed that triuret:G RNA base pairs closely resemble U:G wobble base pairs. Based on the probable abundance of urea on the early Earth, we postulate that urea-containing RNA bases are good candidates for a proto-RNA world., (© 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Biological information is most commonly thought of in terms of biology's Central Dogma where DNA is viewed as a linearized code used to synthesize proteins. Using DNA's chemical cousin, RNA, as a case study we consider how biological information operates outside the linear arrangement of its polymeric subunits. Much like individual pieces of a jigsaw puzzle, particular structures enable biomolecules to undergo precise molecular interactions with one another based on their respective shapes. By exploring the relationship between sequence and structure in RNA we argue that biological information finds its ultimate functional fulfillment in the three-dimensional structural arrangement of its atoms. We show how recurrent structural RNA motifs-operating at the tertiary level of a molecule-provide robust building blocks for the formation of new structural configurations and thereby convey the information required for emergent biological functions. We posit that these same RNA structures, guided by their respective thermodynamic stabilities, experience selective pressure to maintain particular three-dimensional architectures over and above pressures to maintain a particular sequence of nucleotides. Ultimately, this framework for understanding the nature of biological information provides a useful paradigm for understanding its origins and how biological information can result from chaotic prebiotic conditions., (Copyright © 2019 Elsevier B.V. All rights reserved.)