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Our current understanding of the chemistry of the primordial genetic material is fragmentary at best. The chemical replication of oligonucleotides long enough to perform catalytic functions is particularly problematic because of the low efficiency of nonenzymatic template copying. Here we show that this problem can be circumvented by assembling a functional ribozyme by the templated ligation of short oligonucleotides. However, this approach creates a new problem because the splint oligonucleotides used to drive ribozyme assembly strongly inhibit the resulting ribozyme. We explored three approaches to the design of splint oligonucleotides that enable efficient ligation but which allow the assembled ribozyme to remain active. DNA splints, splints with G:U wobble pairs, and splints with G to I (Inosine) substitutions all allowed for the efficient assembly of an active ribozyme ligase. Our work demonstrates the possibility of a transition from nonenzymatic ligation to enzymatic ligation and reveals the importance of avoiding ribozyme inhibition by complementary oligonucleotides.

The RNA world hypothesis describes a scenario where early life forms relied on RNA to govern both inheritance and catalyze useful chemical reactions. Prior to the emergence of enzymes capable of replicating the RNA genome, a nonenzymatic replication process would have been necessary to initiate Darwinian Evolution. However, the one-pot nonenzymatic RNA chemical copying of templates with mixed-sequences is insufficient to generate strand products long enough to encode useful function. The use of alternate (RNA-like) genetic polymers may overcome hurdles associated with RNA copying, and further our understanding of nonenzymatic copying chemistry. This protocol describes the nonenzymatic copying of RNA templates into N3'→P5' phosphoramidate DNA (3'-NP-DNA). We describe, in detail, the synthesis of 3'-amino-2',3'-dideoxyribonucleotide monomers activated with 2-aminoimidazole (3'-NH 2 -2AIpddN), and their use in template-directed polymerization., Competing Interests: Competing interestsThe authors declare no competing financial interest., (Copyright © 2020 The Authors; exclusive licensee Bio-protocol LLC.)

The nonenzymatic replication of ribonucleic acid (RNA) may have enabled the propagation of genetic information during the origin of life. RNA copying can be initiated in the laboratory with chemically activated nucleotides, but continued copying requires a source of chemical energy for in situ nucleotide activation. Recent work has illuminated a potentially prebiotic cyanosulfidic chemistry that activates nucleotides, but its application to nonenzymatic RNA copying had not been demonstrated. Here, we report a novel pathway that activates RNA nucleotides in a manner compatible with template-directed nonenzymatic copying. We show that this pathway, which we refer to as bridge-forming activation, selectively yields the reactive imidazolium-bridged dinucleotide intermediate required for copying. Our results will enable more realistic simulations of RNA propagation based on continuous in situ nucleotide activation.

The non-enzymatic replication of the primordial genetic material is thought to have enabled the evolution of early forms of RNA-based life. However, the replication of oligonucleotides long enough to encode catalytic functions is problematic due to the low efficiency of template copying with mononucleotides. We show that template-directed ligation can assemble long RNAs from shorter oligonucleotides, which would be easier to replicate. The rate of ligation can be greatly enhanced by employing a 3'-amino group at the 3'-end of each oligonucleotide, in combination with an N-alkyl imidazole organocatalyst. These modifications enable the copying of RNA templates by the multistep ligation of tetranucleotide building blocks, as well as the assembly of long oligonucleotides using short splint oligonucleotides. We also demonstrate the formation of long oligonucleotides inside model prebiotic vesicles, which suggests a potential route to the assembly of artificial cells capable of evolution., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Life emerging in an RNA world is expected to propagate RNA as hereditary information, requiring some form of primitive replication without enzymes. Non-enzymatic template-directed RNA primer extension is a model of the copying step in this posited form of replication. The sequence space accessed by primer extension dictates potential pathways to self-replication and, eventually, ribozymes. Which sequences can be accessed? What is the fidelity of the reaction? Does the recently illuminated mechanism of primer extension affect the distribution of sequences that can be copied? How do sequence features respond to experimental conditions and prebiotically relevant contexts? To help answer these and related questions, we here introduce a deep-sequencing methodology for studying RNA primer extension. We have designed and vetted special RNA constructs for this purpose, honed a protocol for sample preparation and developed custom software that analyzes sequencing data. We apply this new methodology to proof-of-concept controls, and demonstrate that it works as expected and reports on key features of the sequences accessed by primer extension., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

A previously proposed synthesis of pyrimidine ribonucleotides makes use of ultraviolet (UV) light to convert β-d-ribocytidine-2',3'-cyclic phosphate to β-d-ribouridine-2',3'-cyclic phosphate, while simultaneously selectively degrading synthetic byproducts. Past studies of the photochemical reactions of pyrimidines have employed mercury arc lamps, characterized by narrowband emission centered at 254 nm, which is not representative of the UV environment of the early Earth. To further assess this process under more realistic circumstances, we investigated the wavelength dependence of the UV-driven conversion of β-d-ribocytidine-2',3'-cyclic phosphate to β-d-ribouridine-2',3'-cyclic phosphate. We used constraints provided by planetary environments to assess the implications for pyrimidine nucleotides on the early Earth. We found that the wavelengths of light (255-285 nm) that most efficiently drive the deamination of β-d-ribocytidine-2',3'-cyclic phosphate to β-d-ribouridine-2',3'-cyclic phosphate are accessible on planetary surfaces such as those of the Hadean-Archaean Earth for CO 2 -N 2 -dominated atmospheres. However, continued irradiation could eventually lead to low levels of ribocytidine in a low-temperature, highly irradiated environment, if production rates are slow.

Many proteins that are needed for progression through S-phase are produced from transcripts that peak in the S-phase, linking temporal expression of those proteins to the time that they are required in cell cycle. Here, we explore the potential roles of long non-coding RNAs in cell cycle progression. We use a sensitive click-chemistry approach to isolate nascent RNAs in a human cell line, and we identify more than 900 long non-coding RNAs (lncRNAs) whose synthesis peaks during the S-phase. More than 200 of these are long intergenic non-coding RNAs (lincRNAs) with S-phase-specific expression. We characterize three of these lincRNAs by knockdown and find that all three lincRNAs are required for appropriate S-phase progression. We infer that non-coding RNAs are key regulatory effectors during the cell cycle, acting on distinct regulatory networks, and herein, we provide a large catalog of candidate cell-cycle regulatory RNAs., Competing Interests: Declaration of Interests The authors declare no competing interests, (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

All known polymerases copy genetic material by catalyzing phosphodiester bond formation. This highly conserved activity proceeds by a common mechanism, such that incorporated nucleoside analogs terminate chain elongation if the resulting primer strand lacks a terminal hydroxyl group. Even conservatively substituted 3'-amino nucleotides generally act as chain terminators, and no enzymatic pathway for their polymerization has yet been found. Although 3'-amino nucleotides can be chemically coupled to yield stable oligonucleotides containing N3'→P5' phosphoramidate (NP) bonds, no such internucleotide linkages are known to occur in nature. Here, we report that 3'-amino terminated primers are, in fact, slowly extended by the DNA polymerase from B. stearothermophilus in a template-directed manner. When its cofactor is Ca 2+ rather than Mg 2+ , the reaction is fivefold faster, permitting multiple turnover NP bond formation to yield NP-DNA strands from the corresponding 3'-amino-2',3'-dideoxynucleoside 5'-triphosphates. A single active site mutation further enhances the rate of NP-DNA synthesis by an additional 21-fold. We show that DNA-dependent NP-DNA polymerase activity depends on conserved active site residues and propose a likely mechanism for this activity based on a series of crystal structures of bound complexes. Our results significantly broaden the catalytic scope of polymerase activity and suggest the feasibility of a genetic transition between native nucleic acids and NP-DNA., Competing Interests: Competing interest statement: The authors have filed for a patent related to this work., (Copyright © 2020 the Author(s). Published by PNAS.)

The hypothesized central role of RNA in the origin of life suggests that RNA propagation predated the advent of complex protein enzymes. A critical step of RNA replication is the template-directed synthesis of a complementary strand. Two experimental approaches have been extensively explored in the pursuit of demonstrating protein-free RNA synthesis: template-directed nonenzymatic RNA polymerization using intrinsically reactive monomers and ribozyme-catalyzed polymerization using more stable substrates such as biological 5'-triphosphates. Despite significant progress in both approaches in recent years, the assembly and copying of functional RNA sequences under prebiotic conditions remains a challenge. Here, we explore an alternative approach to RNA-templated RNA copying that combines ribozyme catalysis with RNA substrates activated with a prebiotically plausible leaving group, 2-aminoimidazole (2AI). We applied in vitro selection to identify ligase ribozymes that catalyze phosphodiester bond formation between a template-bound primer and a phosphor-imidazolide-activated oligomer. Sequencing revealed the progressive enrichment of 10 abundant sequences from a random sequence pool. Ligase activity was detected in all 10 RNA sequences; all required activation of the ligator with 2AI and generated a 3'-5' phosphodiester bond. We propose that ribozyme catalysis of phosphodiester bond formation using intrinsically reactive RNA substrates, such as imidazolides, could have been an evolutionary step connecting purely nonenzymatic to ribozyme-catalyzed RNA template copying during the origin of life., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

The abiotic synthesis of ribonucleotides is thought to have been an essential step toward the emergence of the RNA world. However, it is likely that the prebiotic synthesis of ribonucleotides was accompanied by the simultaneous synthesis of arabinonucleotides, 2'-deoxyribonucleotides, and other variations on the canonical nucleotides. In order to understand how relatively homogeneous RNA could have emerged from such complex mixtures, we have examined the properties of arabinonucleotides and 2'-deoxyribonucleotides in nonenzymatic template-directed primer extension reactions. We show that nonenzymatic primer extension with activated arabinonucleotides is much less efficient than with activated ribonucleotides, and furthermore that once an arabinonucleotide is incorporated, continued primer extension is strongly inhibited. As previously shown, 2'-deoxyribonucleotides are also less efficiently incorporated in primer extension reactions, but the difference is more modest. Experiments with mixtures of nucleotides suggest that the coexistence of ribo- and arabinonucleotides does not impede the copying of RNA templates. Moreover, chimeric oligoribonucleotides containing 2'-deoxy- or arabinonucleotides are effective templates for RNA synthesis. We propose that the initial genetic polymers were random sequence chimeric oligonucleotides formed by untemplated polymerization, but that template copying chemistry favored RNA synthesis; multiple rounds of replication may have led to pools of oligomers composed mainly of RNA.

Laboratory evolution of functional RNAs has applications in many areas of chemical and synthetic biology. In vitro selections critically depend on the presence of functional molecules, such as aptamers and ribozymes, in the starting sequence pools. For selection of novel functions the pools are typically transcribed from random-sequence DNA templates, yielding a highly diverse set of RNAs that contain a multitude of folds and biochemical activities. The phenotypic potential, the frequency of functional RNAs, is very low, requiring large complexity of starting pools, surpassing 10 15 different sequences, to identify highly active isolates. Furthermore, the majority of random sequences is not structured and has a high propensity for aggregation; the in vitro selection process thus involves not just enrichment of functional RNAs, but also their purification from aggregation-prone "free-riders". We reasoned that purification of the nonaggregating, monomeric subpopulation of a random-sequence RNA pool will yield pools of folded, functional RNAs. We performed six rounds of selection for monomeric sequences and show that the enriched population is compactly folded. In vitro selections originating from various mixtures of the compact pool and a fully random pool showed that sequences from the compact pool always dominate the population once a biochemical activity is detectable. A head-to-head competition of the two pools starting from a low (5 × 10 12 ) sequence diversity revealed that the phenotypic potential of the compact pool is about 1000-times higher than the fully random pool. A selection for folded and monomeric RNA pools thus greatly increases the frequency of functional RNAs from that seen in random-sequence pools, providing a facile experimental approach to isolation of highly active functional RNAs from low-diversity populations.

Achieving multiple cycles of RNA replication within a model protocell would be a critical step toward demonstrating a path from prebiotic chemistry to cellular biology. Any model for early life based on an "RNA world" must account for RNA strand cleavage and hydrolysis, which would degrade primitive genetic information and lead to an accumulation of truncated, phosphate-terminated strands. We show here that cleavage of the phosphodiester backbone is not an end point for RNA replication. Instead, 3'-phosphate-terminated RNA strands can participate in template-directed copying reactions with activated ribonucleotide monomers. These reactions form a pyrophosphate linkage, the stability of which we have characterized in the context of RNA copying chemistry. The presence of free magnesium cations results in cleavage of the pyrophosphate bond within minutes. However, we found that the pyrophosphate bond is relatively stable within an RNA duplex and in the presence of chelated magnesium. We show that, under these conditions, pyrophosphate-linked RNA can act as a template for the polymerization of ribonucleotides into canonical 3'-5' phosphodiester-linked RNA. We suggest that primer extension of 3'-phosphate-terminated RNA followed by template-directed copying represents a plausible nonenzymatic pathway for the salvage and recovery of genetic information following strand cleavage.

Primitive life must have possessed the essential features of modern cellular life, but without highly evolved proteins to perform dynamic functions such as nutrient transport and membrane remodeling. Here, we consider the membrane properties of protocells - minimal cells with hereditary material, capable of growth and division - and how these properties place restrictions on the components of the membrane. For example, the lipids of modern membranes are diacyl amphiphilic molecules containing well-over 20 carbons in total. Without proteins, these membranes are very stable and kinetically trapped. This inertness, combined with the need for enzymes to synthesize them, makes modern diacyl amphiphiles unsuitable candidates for the earliest membranes on Earth. We, therefore, discuss the progress made thus far with single-chained amphiphiles, including fatty acids and mixtures of fatty acids with related molecules, and the membrane-related research that must be undertaken to gain more insight into the origins of cellular life., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society and the Royal Society of Biology.)

Non-enzymatic RNA self-replication is integral to the emergence of the 'RNA World'. Despite considerable progress in non-enzymatic template copying, demonstrating a full replication cycle remains challenging due to the difficulty of separating the strands of the product duplex. Here, we report a prebiotically plausible approach to strand displacement synthesis in which short 'invader' oligonucleotides unwind an RNA duplex through a toehold/branch migration mechanism, allowing non-enzymatic primer extension on a template that was previously occupied by its complementary strand. Kinetic studies of single-step reactions suggest that following invader binding, branch migration results in a 2:3 partition of the template between open and closed states. Finally, we demonstrate continued primer extension with strand displacement by employing activated 3'-aminonucleotides, a more reactive proxy for ribonucleotides. Our study suggests that complete cycles of non-enzymatic replication of the primordial genetic material may have been facilitated by short RNA oligonucleotides., Competing Interests: LZ, SK, KH, DO, CG, TW, JS No competing interests declared, (© 2019, Zhou et al.)

Genetic polymers that could plausibly govern life in the universe might inhabit a broad swath of chemical space. A subset of these genetic systems can exchange information with RNA and DNA and could therefore form the basis for model protocells in the laboratory. N3'→P5' phosphoramidate (NP) DNA is defined by a conservative linkage substitution and has shown promise as a protocellular genetic material, but much remains unknown about its functionality and fidelity due to limited enzymatic tools. Conveniently, we find widespread NP-DNA-dependent DNA polymerase activity among reverse transcriptases, an observation consistent with structural studies of the RNA-like conformation of NP-DNA duplexes. Here, we analyze the consequences of this unnatural template linkage on the kinetics and fidelity of DNA polymerization activity catalyzed by wild-type and variant reverse transcriptases. Template-associated deficits in kinetics and fidelity suggest that even highly conservative template modifications give rise to error-prone DNA polymerase activity. Enzymatic copying of NP-DNA sequences is nevertheless an important step toward the future study and engineering of this synthetic genetic polymer., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Three related molecules in the 2-aminoazole family are potentially important for prebiotic chemistry: 2-aminooxazole, 2-aminoimidazole, and 2-aminothiazole, which can provide critical functions as an intermediate in nucleotide synthesis, a nucleotide activating agent, and a selective agent, respectively. Here, we examine the wavelength-dependent photodegradation of these three molecules under mid-range UV light (210-290 nm). We then assess the implications of the observed degradation rates for the proposed prebiotic roles of these compounds. We find that all three 2-aminoazoles degrade under UV light, with half lives ranging from ≈7-100 hours under a solar-like spectrum. 2-Aminooxazole is the least photostable, while 2-aminoimidazole is the most photostable. The relative photostabilities are consistent with the order in which these molecules would be used prebiotically: AO is used first to build nucleotides and AI is used last to activate them.

The emergence of the replication of RNA oligonucleotides was a critical step in the origin of life. An important model for the study of nonenzymatic template copying, which would be a key part of any such pathway, involves the reaction of ribonucleoside-5'-phosphorimidazolides with an RNA primer/template complex. The mechanism by which the primer becomes extended by one nucleotide was assumed to be a classical in-line nucleophilic-substitution reaction in which the 3'-hydroxyl of the primer attacks the phosphate of the incoming activated monomer with displacement of the imidazole leaving group. Surprisingly, this simple model has turned out to be incorrect, and the dominant pathway has now been shown to involve the reaction of two activated nucleotides with each other to form a 5'-5'-imidazolium bridged dinucleotide intermediate. Here we review the discovery of this unexpected intermediate, and the chemical, kinetic, and structural evidence for its role in template copying chemistry., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Efforts to develop self-replicating nucleic acids have led to insights into the origin of life and have also suggested potential pathways to the design of artificial life forms based on non-natural nucleic acids. The template-directed nonenzymatic polymerization of activated ribonucleotide monomers is generally slow because of the relatively weak nucleophilicity of the primer 3'-hydroxyl. To circumvent this problem, several nucleic acids based on amino-sugar nucleotides have been studied, and as expected, the more-nucleophilic amine generally results in faster primer extension. Extending this logic, we have chosen to study morpholino nucleic acid (MoNA), because the secondary amine of the morpholino-nucleotides is expected to be highly nucleophilic. We describe the synthesis of 2-methylimidazole-activated MoNA monomers from their corresponding ribonucleoside 5'-monophosphates and the synthesis of an RNA primer with a terminal MoNA nucleotide. We show that the activated G and C MoNA monomers enable rapid and efficient extension of the morpholino-terminated primer on homopolymeric and mixed-sequenced RNA templates. Our results show that MoNA is a non-natural informational polymer that is worthy of further study as a candidate self-replicating material.

Efficiently copying mixed-sequence oligonucleotide templates nonenzymatically is a long-standing problem both with respect to the origin of life, and with regard to bottom up efforts to synthesize artificial living systems. Here we report an efficient and sequence-general nonenzymatic process in which RNA templates direct the synthesis of a complementary strand composed of N3'→P5' phosphoramidate DNA (3'-NP-DNA) using 3'-amino-2',3'-dideoxyribonucleotides activated with 2-aminoimidazole. Using only the four canonical nucleobases (A, G, C, and T) of modern DNA, we demonstrate the chemical copying of a variety of mixed-sequence RNA templates, both in solution and within model protocells, into complementary 3'-NP-DNA strands. Templates up to 25 nucleotides long were chemically transcribed with an average stepwise yield of 96-97%. The nonenzymatic template-directed generation of primer extension products long enough to encode active ribozymes and/or aptamers inside model protocells suggests possible routes to the synthesis of evolving cellular systems.

The ability of the biopolymers RNA and DNA to store and transfer information is essential to life. Herein, we demonstrate template-directed replication in a set of dimer duplexes that use reversible covalent bonds to form base-pairing interactions. Binary sequence information was encoded as a sequence of aniline and benzaldehyde subunits linked together by a diethynyl benzene backbone. These dimers formed sequence-specific, imine-linked duplexes, which could be separated and used as templates for the synthesis of daughter duplexes with identical sequences.

Having a fast, reliable method for characterizing vesicles is vital for their use as model cell membranes in biophysics, synthetic biology, and origins of life studies. Instead of the traditionally used Rayleigh-Gans-Debye approximation, we use an exact extended Lorenz-Mie solution for how core-shell particles scatter light to model vesicle turbidity. This approach enables accurate interpretations of simple turbidimetric measurements and is able to accurately model highly scattering vesicles, such as larger vesicles, those with multiple layers, and those with encapsulated material. We uncover several surprising features, including that vesicle lamellarity has a larger effect on sample turbidity than vesicle size and that the technique can be used to measure the membrane thickness of vesicles. We also examine potential misinterpretations of turbidimetry and discuss when measurements are limited by forward and multiple scattering and by the geometry of the instrument., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Before the advent of polymerase enzymes, the copying of genetic material during the origin of life may have involved the nonenzymatic polymerization of RNA monomers that are more reactive than the biological nucleoside triphosphates. Activated RNA monomers such as nucleotide 5'-phosphoro-2-aminoimidazolides spontaneously form an imidazolium-bridged dinucleotide intermediate that undergoes rapid nonenzymatic template-directed primer extension. However, it is unknown whether the intermediate can form on the template or only in solution and whether the intermediate is prone to hydrolysis when bound to the template or reacts preferentially with the primer. Here we show that an activated monomer can first bind the template and then form an imidazolium-bridged intermediate by reacting with a 2-aminoimidazole-activated downstream oligonucleotide. We have also characterized the partition of the template-bound intermediate between hydrolysis and primer extension. In the presence of the catalytic metal ion Mg 2+ , >90% of the template-bound intermediate reacts with the adjacent primer to generate the primer extension product while less than 10% reacts with competing water. Our results indicate that an RNA template can catalyze a multistep phosphodiester bond formation pathway while minimizing hydrolysis with a specificity reminiscent of an enzyme-catalyzed reaction.

Many biologists, biochemists, and biophysicists study giant vesicles, which have a diameter of >1 μm, owing to their ease of characterization using standard optical methods. More recently, there has been interest in using giant vesicles as model systems for living cells and for the construction of artificial cells. In fact, there have been a number of reports about functionalizing giant vesicles using membrane-bound pore proteins and encapsulating biochemical reactions. Among the various methods for preparing giant vesicles, the water-in-oil emulsion transfer method is particularly well established. However, the giant vesicles prepared by this method have complex and heterogeneous properties, such as particle size and membrane structure. Here, we demonstrate the characterization of giant vesicles by imaging flow cytometry to provide quantitative and qualitative information about the vesicle products prepared by the water-in-oil emulsion transfer method. Through image-based analyses, several kinds of protocol byproducts, such as oil droplets and vesicles encapsulating no target molecules, were identified and successfully quantified. Further, the optimal agitation conditions for the water-in-oil emulsion transfer method were found from detailed analysis of imaging flow cytometry data. Our results indicate that a sonication-based water-in-oil emulsion transfer method exhibited a higher efficiency in producing giant vesicles, about 10 times or higher than that of vortex and rumble strip-based methods. It is anticipated that these approaches will be useful for fine-tuning giant vesicle production and subsequent applications.

The emergence of primordial RNA-based life would have required the abiotic synthesis of nucleotides, and their participation in nonenzymatic RNA replication. Although considerable progress has been made toward potentially prebiotic syntheses of the pyrimidine nucleotides (C and U) and their 2-thio variants, efficient routes to the canonical purine nucleotides (A and G) remain elusive. Reported syntheses are low yielding and generate a large number of undesired side products. Recently, a potentially prebiotic pathway to 8-oxo-adenosine and 8-oxo-inosine has been demonstrated, raising the question of the suitability of the 8-oxo-purines as substrates for prebiotic RNA replication. Here we show that the 8-oxo-purine nucleotides are poor substrates for nonenzymatic RNA primer extension, both as activated monomers and when present in the template strand; their presence at the end of a primer also strongly reduces the rate and fidelity of primer extension. To provide a proper comparison with 8-oxo-inosine, we also examined primer extension reactions with inosine, and found that inosine exhibits surprisingly rapid and accurate nonenzymatic RNA copying. We propose that inosine, which can be derived from adenosine by deamination, could have acted as a surrogate for G in the earliest stages of the emergence of life., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

The study of nonenzymatic template-directed RNA copying is the experimental basis for the search for chemistry and reaction conditions consistent with prebiotic RNA replication. The most effective model systems for RNA copying have to date required a high concentration of Mg 2+ . Recently, Fe 2+ , which was abundant on the prebiotic anoxic Earth, was shown to promote the folding of RNA in a manner similar to the case of Mg 2+ , as a result of the two cations having similar interactions with phosphate groups. These observations raise the question of whether Fe 2+ could have promoted RNA copying on the prebiotic Earth. Here, we demonstrate that Fe 2+ is a better catalyst and promotes faster nonenzymatic RNA primer extension and ligation than Mg 2+ when using 2-methylimidazole activated nucleotides in slightly acidic to neutral pH solutions. Thus, it appears likely that Fe 2+ could have facilitated RNA replication and evolution in concert with other metal cations on the prebiotic Earth.

The general notion of an "RNA world" is that, in the early development of life on the Earth, genetic continuity was assured by the replication of RNA, and RNA molecules were the chief agents of catalytic function. Assuming that all of the components of RNA were available in some prebiotic locale, these components could have assembled into activated nucleotides that condensed to form RNA polymers, setting the stage for the chemical replication of polynucleotides through RNA-templated RNA polymerization. If a sufficient diversity of RNAs could be copied with reasonable rate and fidelity, then Darwinian evolution would begin with RNAs that facilitated their own reproduction enjoying a selective advantage. The concept of a "protocell" refers to a compartment where replication of the primitive genetic material took place and where primitive catalysts gave rise to products that accumulated locally for the benefit of the replicating cellular entity. Replication of both the protocell and its encapsulated genetic material would have enabled natural selection to operate based on the differential fitness of competing cellular entities, ultimately giving rise to modern cellular life., (Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.)

Non-enzymatic RNA replication may have been one of the processes involved in the appearance of life on Earth. Attempts to recreate this process in a laboratory setting have not been successful thus far, highlighting a critical need for finding prebiotic conditions that increase the rate and the yield. Now a highly parallel assay for template directed RNA synthesis is presented that relies on the intrinsic fluorescence of a 2-aminopurine modified G-quadruplex. The application of the assay to examine the combined influence of multiple variables including pH, divalent metal concentrations and ribonucleotide concentrations on the copying of RNA sequences is demonstrated. The assay enables a direct survey of physical and chemical conditions, potentially prebiotic, which could enable the chemical replication of RNA., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

tRNAs are subject to numerous modifications, including methylation. Mutations in the human N 7 -methylguanosine (m 7 G) methyltransferase complex METTL1/WDR4 cause primordial dwarfism and brain malformation, yet the molecular and cellular function in mammals is not well understood. We developed m 7 G methylated tRNA immunoprecipitation sequencing (MeRIP-seq) and tRNA reduction and cleavage sequencing (TRAC-seq) to reveal the m 7 G tRNA methylome in mouse embryonic stem cells (mESCs). A subset of 22 tRNAs is modified at a "RAGGU" motif within the variable loop. We observe increased ribosome occupancy at the corresponding codons in Mettl1 knockout mESCs, implying widespread effects on tRNA function, ribosome pausing, and mRNA translation. Translation of cell cycle genes and those associated with brain abnormalities is particularly affected. Mettl1 or Wdr4 knockout mESCs display defective self-renewal and neural differentiation. Our study uncovers the complexity of the mammalian m 7 G tRNA methylome and highlights its essential role in ESCs with links to human disease., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The importance of genome replication has inspired detailed crystallographic studies of enzymatic DNA/RNA polymerization. In contrast, the mechanism of nonenzymatic polymerization is less well understood, despite its critical role in the origin of life. Here we report the direct observation of nonenzymatic RNA primer extension through time-resolved crystallography. We soaked crystals of an RNA primer-template-dGMP complex with guanosine-5'-phosphoro-2-aminoimidazolide for increasing times. At early times we see the activated ribonucleotides bound to the template, followed by formation of the imidazolium-bridged dinucleotide intermediate. At later times, we see a new phosphodiester bond forming between the primer and the incoming nucleotide. The intermediate is pre-organized because of the constraints of base-pairing with the template and hydrogen bonding between the imidazole amino group and both flanking phosphates. Our results provide atomic-resolution insight into the mechanism of nonenzymatic primer extension, and set the stage for further structural dissection and optimization of the RNA copying process., Competing Interests: WZ, TW, LL, JS No competing interests declared, (© 2018, Zhang et al.)

The chemical replication of RNA inside fatty acid vesicles is a plausible step in the emergence of cellular life. On the primitive Earth, simple protocells with the ability to import nucleotides and short oligomers from their environment could potentially have replicated and retained larger genomic RNA oligonucleotides within a spatially defined compartment. We have previously shown that short 5'-phosphoroimidazolide-activated "helper" RNA oligomers enable the nonenzymatic copying of mixed-sequence templates in solution, using 5'-phosphoroimidazolide-activated mononucleotides. Here, we report that citrate-chelated Mg 2+ , a catalyst of nonenzymatic primer extension, enhances fatty acid membrane permeability to such short RNA oligomers up to the size of tetramers, without disrupting vesicle membranes. In addition, selective permeability of short, but not long, oligomers can be further enhanced by elevating the temperature. The ability to increase the permeability of fatty acid membranes to short oligonucleotides allows for the nonenzymatic copying of RNA templates containing all four nucleotides inside vesicles, bringing us one step closer to the goal of building a protocell capable of Darwinian evolution.

Prior to the evolution of membrane proteins, intrinsic membrane stability and permeability to polar solutes are essential features of a primitive cell membrane. These features are difficult to achieve simultaneously in model protocells made of either pure fatty acid or phospholipid membranes, raising the intriguing question of how the transition from fatty acid to phospholipid membranes might have occurred while continuously supporting encapsulated reactions required for genomic replication. Here, the properties of a blended membrane system composed of both oleic acid (OA), a monoacyl fatty acid, and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), a diacyl phospholipid are described. This hybrid vesicle system exhibits high stability to divalent cations (Mg 2+ ), while simultaneously maintaining its permeability to small charged molecules such as nucleotides and divalent ions such as Mg 2+ . This combination of features facilitates key reactions expected to occur during a transition from primitive to modern cells, including nonenzymatic RNA replication, and is also compatible with highly evolved functions such as the ribosomal translation of a protein. The observations support the hypothesis that the early transition from fatty acid to phospholipid membranes could be accomplished through intermediate states in which membranes are composed of amphiphile mixtures, and do not require protein transporters., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Nonenzymatic RNA primer extension by activated mononucleotides has long served as a model for the study of prebiotic RNA copying. We have recently shown that the rate of primer extension is greatly enhanced by the formation of an imidazolium-bridged dinucleotide between the incoming monomer and a second, downstream activated monomer. However, the rate of primer extension is further enhanced if the downstream monomer is replaced by an activated oligonucleotide. Even an unactivated downstream oligonucleotide provides a modest enhancement in the rate of reaction of a primer with a single activated monomer. Here we study the mechanism of these effects through crystallographic studies of RNA complexes with the recently synthesized nonhydrolyzable substrate analog, guanosine 5'-(4-methylimidazolyl)-phosphonate (ICG). ICG mimics 2-methylimidazole activated guanosine-5'-phosphate (2-MeImpG), a commonly used substrate in nonenzymatic primer extension experiments. We present crystal structures of primer-template complexes with either one or two ICG residues bound downstream of a primer. In both cases, the aryl-phosphonate moiety of the ICG adjacent to the primer is disordered. To investigate the effect of a downstream oligonucleotide, we transcribed a short RNA oligonucleotide with either a 5'-ICG residue, a 5'-phosphate or a 5'-hydroxyl. We then determined crystal structures of primer-template complexes with a bound ICG monomer sandwiched between the primer and each of the three downstream oligonucleotides. Surprisingly, all three oligonucleotides rigidify the ICG monomer conformation and position it for attack by the primer 3'-hydroxyl. Furthermore, when GpppG, an analog of the imidazolium-bridged intermediate, is sandwiched between an upstream primer and a downstream helper oligonucleotide, or covalently linked to the 5'-end of the downstream oligonucleotide, the complex is better preorganized for primer extension than in the absence of a downstream oligonucleotide. Our results suggest that a downstream helper oligonucleotide contributes to the catalysis of primer extension by favoring a reactive conformation of the primer-template-intermediate complex.

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of these amphiphiles. In particular, fatty acid liposomes enhance dynamic character, due to the relatively high solubility of single-chain amphiphiles. Similarly, liposomes containing free fatty acids are more sensitive to salt and divalent cations, due to the strong interactions between the carboxylic acid head groups and metal ions. Here we illustrate techniques for preparation, purification, and use of liposomes comprised in part or whole of single chain amphiphiles (e.g., oleic acids).

UV-driven photoredox processing of cyanocuprates can generate simple sugars necessary for prebiotic synthesis. We investigate the wavelength dependence of this process from 215 to 295 nm and generally observe faster rates at shorter wavelengths. The most efficient wavelengths are accessible to a range of potential prebiotic atmospheres, supporting the potential role of cyanocuprate photochemistry in prebiotic synthesis on the early Earth.

We report the synthesis of guanosine 5'-(4-methylimidazolyl)phosphonate (ICG), the third member of a series of nonhydrolyzable nucleoside 5'-phosphoro-2-methylimidazolide (2-MeImpN) analogues designed for mechanistic studies of nonenzymatic RNA primer extension. The addition of a 2-MeImpN monomer to a primer is catalyzed by the presence of a downstream activated monomer, yet the three nonhydrolyzable analogues do not show catalytic effects under standard mildly basic primer extension conditions. Surprisingly, ICG, which has a pK a similar to that of 2-MeImpG, is a modest catalyst of nonenzymatic primer extension at acidic pH. Here we show that ICG reacts with 2-MeImpC to form a stable 5'-5'-imidazole-bridged guanosine-cytosine dinucleotide, with both a labile nitrogen-phosphorus and a stable carbon-phosphorus linkage flanking the central imidazole bridge. Cognate RNA primer-template complexes react with this GC-dinucleotide by attack of the primer 3'-hydroxyl on the activated N-P side of the 5'-5'-imidazole bridge. These observations support the hypothesis that 5'-5'-imidazole-bridged dinucleotides can bind to cognate RNA primer-template duplexes and adopt appropriate conformations for subsequent phosphodiester bond formation, consistent with our recent mechanistic proposal that the formation of activated 5'-5'-imidazolium-bridged dinucleotides is responsible for 2-MeImpN-driven primer extension.

It remains a formidable challenge to characterize the diverse complexes of RNA binding proteins and their targets. While crosslink and immunoprecipitation (CLIP) methods are powerful techniques that identify RNA targets on a global scale, the resolution and consistency of these methods is a matter of debate. Here we present a comparative analysis of LIN28-pre-let-7 UV-induced crosslinking using a tandem mass spectrometry (MS/MS) and deep sequencing interrogation of in vitro crosslinked complexes. Interestingly, analyses by the two methods diverge in their identification of crosslinked nucleotide identity - whereas bioinformatics and sequencing analyses suggest guanine in mammalian cells, MS/MS identifies uridine. This work suggests the need for comprehensive analysis and validation of crosslinking methodologies.

Iron-sulfur clusters are ancient cofactors that play a fundamental role in metabolism and may have impacted the prebiotic chemistry that led to life. However, it is unclear whether iron-sulfur clusters could have been synthesized on prebiotic Earth. Dissolved iron on early Earth was predominantly in the reduced ferrous state, but ferrous ions alone cannot form polynuclear iron-sulfur clusters. Similarly, free sulfide may not have been readily available. Here we show that UV light drives the synthesis of [2Fe-2S] and [4Fe-4S] clusters through the photooxidation of ferrous ions and the photolysis of organic thiols. Iron-sulfur clusters coordinate to and are stabilized by a wide range of cysteine-containing peptides and the assembly of iron-sulfur cluster-peptide complexes can take place within model protocells in a process that parallels extant pathways. Our experiments suggest that iron-sulfur clusters may have formed easily on early Earth, facilitating the emergence of an iron-sulfur-cluster-dependent metabolism.

This corrects the article DOI: 10.1038/nchem.2551.

A working model of nonenzymatic RNA primer extension could illuminate how prebiotic chemistry transitioned to biology. All currently known experimental reconstructions of nonenzymatic RNA primer extension yield a mixture of 2'-5' and 3'-5' internucleotide linkages. Although long seen as a major problem, the causes of the poor regioselectivity of the reaction are unknown. We used a combination of different leaving groups, nucleobases, and templating sequences to uncover the factors that yield selective formation of 3'-5' internucleotide linkages. We found that fast and high yielding reactions selectively form 3'-5' linkages. Additionally, in all cases with high 3'-5' regioselectivity, Watson-Crick base pairing between the RNA monomers and the template is observed at the extension site and at the adjacent downstream position. Mismatched base-pairs and other factors that would perturb the geometry of the imidazolium bridged intermediate lower both the rate and regioselectivity of the reaction.

The nonenzymatic polymerization of RNA may have enabled copying of functional sequences during the origin of life. Recent progress utilizing 5'-phosphoro-2-aminoimidazole activation has reinvigorated the possibility of using nonenzymatic RNA polymerization for copying arbitrary sequences. However, the reasons why 2-aminoimidazole (AI) is a superior activation group remain unclear. Here we report that the predominant mechanism of polymerization using cytidine-5'-phosphoro-2-aminoimidazolide (Cp*) involves a 2-aminoimidazolium-bridged dinucleotide (Cp*pC) intermediate. To explore the role of this intermediate, we first identify and quantify four reactions involving the synthesis and breakdown of Cp*pC that occur in the absence of the primer-template duplex. We then analyze the dependence of the rate of polymerization on the concentration of the Cp*pC intermediate in the presence and absence of the competitive inhibitor Cp. We also show that the contribution of the monomer Cp* to the polymerization rate is negligible under our primer extension conditions. Finally, we use the experimentally determined rate constants of these reactions to develop a kinetic model that helps explain the changing rate of nonenzymatic RNA polymerization over time. Our model accounts for the concentration of Cp*pC formed by Cp* under primer extension conditions. The model does not completely account for the decline in polymerization rate observed over long times, which indicates that additional important inhibitory processes have not yet been identified. Our results suggest that the superiority of 2-aminoimidazole over the traditional 2-methylimidazole activation is mostly due to the higher level of accumulation of the imidazolium-bridged intermediate under primer extension conditions.

The sequence of events that gave rise to the first life on our planet took place in the Earth's deep past, seemingly forever beyond our reach. Perhaps for that very reason the idea of reconstructing our ancient story is tantalizing, almost irresistible. Understanding the processes that led to synthesis of the chemical building blocks of biology and the ways in which these molecules self-assembled into cells that could grow, divide and evolve, nurtured by a rich and complex environment, seems at times insurmountably difficult. And yet, to my own surprise, simple experiments have revealed robust processes that could have driven the growth and division of primitive cell membranes. The nonenzymatic replication of RNA is more complicated and less well understood, but here too significant progress has come from surprising developments. Even our efforts to combine replicating compartments and genetic materials into a full protocell model have moved forward in unexpected ways. Fortunately, many challenges remain before we will be close to a full understanding of the origin of life, so the future of research in this field is brighter than ever!, (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The nonenzymatic copying of RNA templates with imidazole-activated nucleotides is a well-studied model for the emergence of RNA self-replication during the origin of life. We have recently discovered that this reaction can proceed through the formation of an imidazolium-bridged dinucleotide intermediate that reacts rapidly with the primer. To gain insight into the relationship between the structure of this intermediate and its reactivity, we cocrystallized an RNA primer-template complex with a close analog of the intermediate, the triphosphate-bridged guanosine dinucleotide GpppG, and solved a high-resolution X-ray structure of the complex. The structure shows that GpppG binds the RNA template through two Watson-Crick base pairs, with the primer 3'-hydroxyl oriented to attack the 5'-phosphate of the adjacent G residue. Thus, the GpppG structure suggests that the bound imidazolium-bridged dinucleotide intermediate would be preorganized to react with the primer by in-line S N 2 substitution. The structures of bound GppG and GppppG suggest that the length and flexibility of the 5'-5' linkage are important for optimal preorganization of the complex, whereas the position of the 5'-phosphate of bound pGpG explains the slow rate of oligonucleotide ligation reactions. Our studies provide a structural interpretation for the observed reactivity of the imidazolium-bridged dinucleotide intermediate in nonenzymatic RNA primer extension., Competing Interests: The authors declare no conflict of interest.

We have recently shown that 2-aminoimidazole is a superior nucleotide activating group for nonenzymatic RNA copying. Here we describe a prebiotic synthesis of 2-aminoimidazole that shares a common mechanistic pathway with that of 2-aminooxazole, a previously described key intermediate in prebiotic nucleotide synthesis. In the presence of glycolaldehyde, cyanamide, phosphate and ammonium ion, both 2-aminoimidazole and 2-aminooxazole are produced, with higher concentrations of ammonium ion and acidic pH favoring the former. Given a 1:1 mixture of 2-aminoimidazole and 2-aminooxazole, glyceraldehyde preferentially reacts and cyclizes with the latter, forming a mixture of pentose aminooxazolines, and leaving free 2-aminoimidazole available for nucleotide activation. The common synthetic origin of 2-aminoimidazole and 2-aminooxazole and their distinct reactivities are suggestive of a reaction network that could lead to both the synthesis of RNA monomers and to their subsequent chemical activation.

Understanding prebiotic nucleotide synthesis is a long standing challenge thought to be essential to elucidating the origins of life on Earth. Recently, remarkable progress has been made, but to date all proposed syntheses account separately for the pyrimidine and purine ribonucleotides; no divergent synthesis from common precursors has been proposed. Moreover, the prebiotic syntheses of pyrimidine and purine nucleotides that have been demonstrated operate under mutually incompatible conditions. Here, we tackle this mutual incompatibility by recognizing that the 8-oxo-purines share an underlying generational parity with the pyrimidine nucleotides. We present a divergent synthesis of pyrimidine and 8-oxo-purine nucleotides starting from a common prebiotic precursor that yields the β-ribo-stereochemistry found in the sugar phosphate backbone of biological nucleic acids. The generational relationship between pyrimidine and 8-oxo-purine nucleotides suggests that 8-oxo-purine ribonucleotides may have played a key role in primordial nucleic acids prior to the emergence of the canonical nucleotides of biology.

Achieving efficient nonenzymatic replication of RNA is an important step toward the synthesis of self-replicating protocells that may mimic early forms of life. Despite recent progress, the nonenzymatic copying of templates containing mixed sequences remains slow and inefficient. Here we demonstrate that activating nucleotides with 2-aminoimidazole results in superior reaction kinetics and improved yields of primer extension reaction products. This new leaving group significantly accelerates monomer addition as well as trimer-assisted RNA primer extension, allowing efficient copying of a variety of short RNA templates with mixed sequences.

Origins of life hypotheses often invoke a transitional phase of nonenzymatic template-directed RNA replication prior to the emergence of ribozyme-catalyzed copying of genetic information. Here, using NMR and ITC, we interrogate the binding affinity of guanosine 5'-monophosphate (GMP) for primer-template complexes when either another GMP, or a helper oligonucleotide, can bind downstream. Binding of GMP to a primer-template complex cannot be significantly enhanced by the possibility of downstream monomer binding, because the affinity of the downstream monomer is weaker than that of the first monomer. Strikingly, GMP binding affinity can be enhanced by ca. 2 orders of magnitude when a helper oligonucleotide is stably bound downstream of the monomer binding site. We compare these thermodynamic parameters to those previously reported for T7 RNA polymerase-mediated replication to help address questions of binding affinity in related nonenzymatic processes.

Many high-resolution crystal structures have contributed to our understanding of the reaction pathway for catalysis by DNA and RNA polymerases, but the structural basis of nonenzymatic template-directed RNA replication has not been studied in comparable detail. Here we present crystallographic studies of the binding of ribonucleotide monomers to RNA primer-template complexes, with the goal of improving our understanding of the mechanism of nonenzymatic RNA copying, and of catalysis by polymerases. To explore how activated ribonucleotides recognize and bind to RNA templates, we synthesized an unreactive phosphonate-linked pyrazole analogue of guanosine 5'-phosphoro-2-methylimidazolide (2-MeImpG), a highly activated nucleotide that has been used extensively to study nonenzymatic primer extension. We cocrystallized this analogue with structurally rigidified RNA primer-template complexes carrying single or multiple monomer binding sites, and obtained high-resolution X-ray structures of these complexes. In addition to Watson-Crick base pairing, we repeatedly observed noncanonical guanine:cytidine base pairs in our crystal structures. In most structures, the phosphate and leaving group moieties of the monomers were highly disordered, while in others the distance from O3' of the primer to the phosphorus of the incoming monomer was too great to allow for reaction. We suggest that these effects significantly influence the rate and fidelity of nonenzymatic RNA replication, and that even primitive ribozyme polymerases could enhance RNA replication by enforcing Watson-Crick base pairing between monomers and primer-template complexes, and by bringing the reactive functional groups into closer proximity.

Early protocells are likely to have arisen from the self-assembly of RNA, peptide, and lipid molecules that were generated and concentrated within geologically favorable environments on the early Earth. The reactivity of these components in a prebiotic environment that supplied sources of chemical energy could have produced additional species with properties favorable to the emergence of protocells. The geochemically plausible activation of amino acids by carbonyl sulfide has been shown to generate short peptides via the formation of cyclic amino acid N-carboxyanhydrides (NCAs). Here, we show that the polymerization of valine-NCA in the presence of fatty acids yields acylated amino acids and peptides via a mixed anhydride intermediate. Notably, N α -oleoylarginine, a product of the reaction between arginine and oleic acid in the presence of valine-NCA, partitions spontaneously into vesicle membranes and mediates the association of RNA with the vesicles. Our results suggest a potential mechanism by which activated amino acids could diversify the chemical functionality of fatty acid membranes and colocalize RNA with vesicles during the formation of early protocells.

The non-enzymatic replication of RNA is thought to have been a critical process required for the origin of life. One unsolved difficulty with non-enzymatic RNA replication is that template-directed copying of RNA results in a double-stranded product. After strand separation, rapid strand reannealing outcompetes slow non-enzymatic template copying, which renders multiple rounds of RNA replication impossible. Here we show that oligoarginine peptides slow the annealing of complementary oligoribonucleotides by up to several thousand-fold; however, short primers and activated monomers can still bind to template strands, and template-directed primer extension can still occur, all within a phase-separated condensed state, or coacervate. Furthermore, we show that within this phase, partial template copying occurs even in the presence of full-length complementary strands. This method to enable further rounds of replication suggests one mechanism by which short non-coded peptides could have enhanced early cellular fitness, and potentially explains how longer coded peptides, that is, proteins, came to prominence in modern biology., Competing Interests: No competing financial interests are declared.

The significance of the precise position of the hydroxyl at the 3'-end of an RNA primer for nonenzymatic template-directed primer extension is not well understood. We show that an RNA primer terminating in 3'-hydroxymethyl-2',3'-dideoxy-guanosine has greatly diminished activity, suggesting that the spatial preorganization of the terminal sugar contributes significantly to the efficiency of primer extension.