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Because of its importance for the origin of life, the nonenzymatic copying of RNA templates has been the subject of intense study for several decades. Previous characterizations of template-directed primer extension using 5'-phosphoryl-2-methylimidazole-activated nucleotides (2-MeImpNs) as substrates have assumed a classical in-line nucleophilic substitution mechanism, in which the 3'-hydroxyl of the primer attacks the phosphate of the incoming monomer, displacing the 2-methylimidazole leaving group. However, we have found that the initial rate of primer extension depends on the pH and concentration at which the activated monomer is maintained prior to the primer extension reaction. These and other results suggest an alternative mechanism, in which two monomers react with each other to form an imidazolium-bridged dinucleotide intermediate, which then binds to the template. Subsequent attack of the 3'-hydroxyl of the primer displaces an activated nucleotide as the leaving group and results in extension of the primer by one nucleotide. Analysis of monomer solutions by NMR indicates formation of the proposed imidazolium-bridged dinucleotide in the expected pH-dependent manner. We have used synthetic methods to prepare material that is enriched in this proposed intermediate and show that it is a highly reactive substrate for primer extension. The formation of an imidazolium-bridged dinucleotide intermediate provides a mechanistic interpretation of previously observed catalysis by an activated nucleotide located downstream from the site of primer extension.

The nonenzymatic replication of RNA is a potential transitional stage between the prebiotic chemistry of nucleotide synthesis and the canonical RNA world in which RNA enzymes (ribozymes) catalyze replication of the RNA genomes of primordial cells. However, the plausibility of nonenzymatic RNA replication is undercut by the lack of a protocell-compatible chemical system capable of copying RNA templates containing all four nucleotides. We show that short 5'-activated oligonucleotides act as catalysts that accelerate primer extension, and allow for the one-pot copying of mixed sequence RNA templates. The fidelity of the primer extension products resulting from the sequential addition of activated monomers, when catalyzed by activated oligomers, is sufficient to sustain a genome long enough to encode active ribozymes. Finally, by immobilizing the primer and template on a bead and adding individual monomers in sequence, we synthesize a significant part of an active hammerhead ribozyme, forging a link between nonenzymatic polymerization and the RNA world.

Ribozyme-catalyzed RNA polymerization is inefficient and error prone. Here we demonstrate that two alternative bases, 2-thio-uridine (s(2)U) and 2-thio-ribo-thymidine (s(2)T), improve the rate and fidelity of ribozyme catalyzed nucleotide addition as NTP substrates and as template bases. We also demonstrate the functionality of s(2)U and s(2)T-containing ribozymes.

The emergence of homeostatic mechanisms that enable maintenance of an intracellular steady state during growth was critical to the advent of cellular life. Here, we show that concentration-dependent reversible binding of short oligonucleotides, of both specific and random sequence, can modulate ribozyme activity. In both cases, catalysis is inhibited at high concentrations, and dilution activates the ribozyme via inhibitor dissociation, thus maintaining near-constant ribozyme specific activity throughout protocell growth. To mimic the result of RNA synthesis within non-growing protocells, we co-encapsulated high concentrations of ribozyme and oligonucleotides within fatty acid vesicles, and ribozyme activity was inhibited. Following vesicle growth, the resulting internal dilution produced ribozyme activation. This simple physical system enables a primitive homeostatic behaviour: the maintenance of constant ribozyme activity per unit volume during protocell volume changes. We suggest that such systems, wherein short oligonucleotides reversibly inhibit functional RNAs, could have preceded sophisticated modern RNA regulatory mechanisms, such as those involving miRNAs.

Phosphoroimidazolides play a critical role in several enzymatic phosphoryl transfer reactions and have been studied extensively as activated monomers for nonenzymatic nucleic acid replication, but the detailed mechanisms of these phosphoryl transfer reactions remain elusive. Some aspects of the mechanism can be deduced by studying the hydrolysis reaction, a simpler system that is amenable to a thorough mechanistic treatment. Here we characterize the transition state of phosphoroimidazolide hydrolysis by kinetic isotope effect (KIE) and linear free energy relationship (LFER) measurements, and theoretical calculations. The KIE and LFER observations are best explained by calculated loose transition structures with extensive scissile bond cleavage. These three-dimensional models of the transition state provide the basis for future mechanistic investigations of phosphoroimidazolide reactions.

One widely held model of early life suggests primitive cells consisted of simple RNA-based catalysts within lipid compartments. One possible selective advantage conferred by an encapsulated catalyst is stabilization of the compartment, resulting from catalyst-promoted synthesis of key membrane components. Here we show model protocell vesicles containing an encapsulated enzyme that promotes the synthesis of simple fatty acid derivatives become stabilized to Mg(2+), which is required for ribozyme activity and RNA synthesis. Thus, protocells capable of such catalytic transformations would have enjoyed a selective advantage over other protocells in high Mg(2+) environments. The synthetic transformation requires both the catalyst and vesicles that solubilize the water-insoluble precursor lipid. We suggest that similar modified lipids could have played a key role in early life, and that primitive lipid membranes and encapsulated catalysts, such as ribozymes, may have acted in conjunction with each other, enabling otherwise-impossible chemical transformations within primordial cells.

We present a scalable synthesis of 3'-amino-3'-deoxy-2-thio-thymidine-5'-phosphoro-2-methylimidazolide, an activated monomer that can copy adenosine residues in nucleic acid templates rapidly without a polymerase. The sulfur atom substitution enhances the rate of template copying by 5-fold compared with the 3'-amino-3'-deoxy-T monomer, while the 3'-amino monomers exhibit a 2- to 30-fold enhancement compared with their ribonucleotide counterparts.

The origin of life is a very rich field, filled with possibilities and ripe for discovery. RNA replication requires chemical energy and vesicle division is easy to do with mechanical energy. These requirements point to a surface lake, perhaps at some time following the period of concentrated cyanide chemistry that gave rise to nucleotides, amino acids and (maybe) fatty acids. A second requirement follows specifically from the nature of the RNA replication cycle, which requires generally cool to moderate temperatures for the copying chemistry, punctuated by brief periods of high temperature for strand separation. Remarkably, lakes in a geothermal active area provide just such a fluctuating temperature environment, because lakes similar to Yellowstone can be generally cool (even ice covered in winter), but they contain numerous hydrothermal vents that emit streams of hot water. Protocells in such an environment would occasionally be swept into these hot water streams, where the transient high temperature exposure would cause RNA strand separation. However, the protocells would be quickly mixed with surrounding cold water, and would therefore cool quickly, before their delicate RNA molecules could be destroyed by heat. Because of the combination of favorable chemical and physical environments, this could be the most likely scenario for the early Earth environment that nurtured the origin of life.

High affinity RNA-protein interactions are critical to cellular function, but directly identifying the determinants of binding within these complexes is often difficult. Here, we introduce a stable isotope mass labeling technique to assign specific interacting nucleotides in an oligonucleotide-protein complex by photo-cross-linking. The method relies on generating site-specific oxygen-18-labeled phosphodiester linkages in oligonucleotides, such that covalent peptide-oligonucleotide cross-link sites arising from ultraviolet irradiation can be assigned to specific sequence positions in both RNA and protein simultaneously by mass spectrometry. Using Lin28A and a let-7 pre-element RNA, we demonstrate that mass labeling permits unambiguous identification of the cross-linked sequence positions in the RNA-protein complex.

Mass spectrometry (MS) is a powerful technique for characterizing noncanonical nucleobases and other chemical modifications in small RNAs, yielding rich chemical information that is complementary to high-throughput indirect sequencing. However, mass spectra are often prohibitively complex when fragment ions are analyzed following either solution phase hydrolysis or gas phase fragmentation. For all but the simplest cases, ions arising from multiple fragmentation events, alternative fragmentation pathways, and diverse salt adducts frequently obscure desired single-cut fragment ions. Here we show that it is possible to take advantage of predictable regularities in liquid chromatographic (LC) separation of optimized RNA digests to greatly simplify the interpretation of complex MS data. A two-dimensional analysis of extracted compound chromatograms permits straightforward and robust de novo sequencing, using a novel Monte Carlo algorithm that automatically generates bidirectional paired-end reads, pinpointing the position of modified nucleotides in a sequence. We demonstrate that these advances permit routine LC-MS sequencing of RNAs containing noncanonical nucleotides, and we furthermore examine the applicability of this approach to the study of oligonucleotides containing artificial modifications as well as those commonly observed in post-transcriptionally modified RNAs.

Cooperative interactions between RNA and vesicle membranes on the prebiotic earth may have led to the emergence of primitive cells. The membrane surface offers a potential platform for the catalysis of reactions involving RNA, but this scenario relies upon the existence of a simple mechanism by which RNA could become associated with protocell membranes. Here, we show that electrostatic interactions provided by short, basic, amphipathic peptides can be harnessed to drive RNA binding to both zwitterionic phospholipid and anionic fatty acid membranes. We show that the association of cationic molecules with phospholipid vesicles can enhance the local positive charge on a membrane and attract RNA polynucleotides. This phenomenon can be reproduced with amphipathic peptides as short as three amino acids. Finally, we show that peptides can cross bilayer membranes to localize encapsulated RNA. This mechanism of polynucleotide confinement could have been important for primitive cellular evolution., (© 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution Non-Commercial NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.)

Nucleobase modifications dramatically alter nucleic acid structure and thermodynamics. 2-thiouridine (s(2)U) is a modified nucleobase found in tRNAs and known to stabilize U:A base pairs and destabilize U:G wobble pairs. The recently reported crystal structures of s(2)U-containing RNA duplexes do not entirely explain the mechanisms responsible for the stabilizing effect of s(2)U or whether this effect is entropic or enthalpic in origin. We present here thermodynamic evaluations of duplex formation using ITC and UV thermal denaturation with RNA duplexes containing internal s(2)U:A and s(2)U:U pairs and their native counterparts. These results indicate that s(2)U stabilizes both duplexes. The stabilizing effect is entropic in origin and likely results from the s(2)U-induced preorganization of the single-stranded RNA prior to hybridization. The same preorganizing effect is likely responsible for structurally resolving the s(2)U:U pair-containing duplex into a single conformation with a well-defined H-bond geometry. We also evaluate the effect of s(2)U on single strand conformation using UV- and CD-monitored thermal denaturation and on nucleoside conformation using (1)H NMR spectroscopy, MD and umbrella sampling. These results provide insights into the effects that nucleobase modification has on RNA structure and thermodynamics and inform efforts toward improving both ribozyme-catalyzed and nonenzymatic RNA copying., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The liposome dialyzer is a small-volume equilibrium dialysis device, built from commercially available materials, that is designed for the rapid exchange of small volumes of an extraliposomal reagent pool against a liposome preparation. The dialyzer is prepared by modification of commercially available dialysis cartridges (Slide-A-Lyzer cassettes), and it consists of a reactor with two 300-μl chambers and a 1.56-cm(2) dialysis surface area. The dialyzer is prepared in three stages: (i) disassembling the dialysis cartridges to obtain the required parts, (ii) assembling the dialyzer and (iii) sealing the dialyzer with epoxy. Preparation of the dialyzer takes ∼1.5 h, not including overnight epoxy curing. Each round of dialysis takes 1-24 h, depending on the analyte and membrane used. We previously used the dialyzer for small-volume non-enzymatic RNA synthesis reactions inside fatty acid vesicles. In this protocol, we demonstrate other applications, including removal of unencapsulated calcein from vesicles, remote loading and vesicle microscopy.

The nonenzymatic replication of primordial RNA is thought to have been a critical step in the origin of life. However, despite decades of effort, the poor rate and fidelity of model template copying reactions have thus far prevented an experimental demonstration of nonenzymatic RNA replication. The overall rate and fidelity of template copying depend, in part, on the affinity of free ribonucleotides to the RNA primer-template complex. We have now used (1)H NMR spectroscopy to directly measure the thermodynamic association constants, Kas, of the standard ribonucleotide monophosphates (rNMPs) to native RNA primer-template complexes. The binding affinities of rNMPs to duplexes with a complementary single-nucleotide overhang follow the order C > G > A > U. Notably, these monomers bind more strongly to RNA primer-template complexes than to the analogous DNA complexes. The relative binding affinities of the rNMPs for complementary RNA primer-template complexes are in good quantitative agreement with the predictions of a nearest-neighbor analysis. With respect to G:U wobble base-pairing, we find that the binding of rGMP to a primer-template complex with a 5'-U overhang is approximately 10-fold weaker than to the complementary 5'-C overhang. We also find that the binding of rGMP is only about 2-fold weaker than the binding of rAMP to 5'-U, consistent with the poor fidelity observed in the nonenzymatic copying of U residues in RNA templates. The accurate Ka measurements for ribonucleotides obtained in this study will be useful for designing higher fidelity, more effective RNA replication systems.

The nonenzymatic replication of RNA oligonucleotides is thought to have played a key role in the origin of life prior to the evolution of ribozyme-catalyzed RNA replication. Although the copying of oligo-C templates by 2-methylimidazole-activated G monomers can be quite efficient, the copying of mixed sequence templates, especially those containing A and U, is particularly slow and error-prone. The greater thermodynamic stability of the 2-thio-U(s(2)U):A base pair, relative to the canonical U:A base pair, suggests that replacing U with s(2)U might enhance the rate and fidelity of the nonenzymatic copying of RNA templates. Here we report that this single atom substitution in the activated monomer improves both the kinetics and the fidelity of nonenzymatic primer extension on mixed-sequence RNA templates. In addition, the mean lengths of primer extension products obtained with s(2)U is greater than those obtained with U, augmenting the potential for nonenzymatic replication of heritable function-rich sequences. We suggest that noncanonical nucleotides such as s(2)U may have played a role during the infancy of the RNA world by facilitating the nonenzymatic replication of genomic RNA oligonucleotides.

The earliest genomic RNAs had to be short enough for efficient replication, while simultaneously serving as unfolded templates and effective ribozymes. A partial solution to this paradox may lie in the fact that many functional RNAs can self-assemble from multiple fragments. Therefore, in early evolution, genomic RNA fragments could have been significantly shorter than unimolecular functional RNAs. Here, we show that unstable, nonfunctional complexes assembled from even shorter 3'-truncated oligonucleotides can be stabilized and gain function via non-enzymatic primer extension. Such short RNAs could act as good templates due to their minimal length and complex-forming capacity, while their minimal length would facilitate replication by relatively inefficient polymerization reactions. These RNAs could also assemble into nascent functional RNAs and undergo conversion to catalytically active forms, by the same polymerization chemistry used for replication that generated the original short RNAs. Such phenomena could have substantially relaxed requirements for copying efficiency in early nonenzymatic replication systems.

The earliest forms of cellular life would have required a membrane compartment capable of growth and division. Fatty acid vesicles are an attractive model of protocell membranes, as they can grow into filamentous vesicles that readily divide while retaining their contents. In order to study vesicle growth, we have developed a method for immobilizing multilamellar fatty acid vesicles on modified glass surfaces and inducing filamentous membrane growth under flow. Filament formation strictly depended on the presence of freshly neutralized fatty acid micelles in the flow chamber. Using light microscopy, we observed a strong dependence of initial growth velocity on initial vesicle size, suggesting that new fatty acid molecules were incorporated into the membrane over the entire external surface of the vesicle. We examined the influences of flow rate, fatty acid concentration, and salt concentration on filamentous growth and observed drastic shape changes, including membrane pearling, of preexisting membrane tubules in response to osmotic stress. These results illustrate the versatility of flow studies for exploring the process of fatty acid vesicle growth following exposure to free fatty acids.

A requirement for concentrated and chemically homogeneous pools of molecular building blocks would severely restrict plausible scenarios for the origin of life. In the case of membrane self-assembly, models of prebiotic lipid synthesis yield primarily short, single-chain amphiphiles that can form bilayer vesicles only at very high concentrations. These high critical aggregation concentrations (cacs) pose significant obstacles for the self-assembly of single-chain lipid membranes. Here, we examine membrane self-assembly in mixtures of fatty acids with varying chain lengths, an expected feature of any abiotic lipid synthesis. We derive theoretical predictions for the cac of mixtures by adapting thermodynamic models developed for the analogous phenomenon of mixed micelle self-assembly. We then use several complementary methods to characterize aggregation experimentally, and find cac values in close agreement with our theoretical predictions. These measurements establish that the cac of fatty acid mixtures is dramatically lowered by minor fractions of long-chain species, thereby providing a plausible route for protocell membrane assembly. Using an NMR-based approach to monitor aggregation of isotopically labeled samples, we demonstrate the incorporation of individual components into mixed vesicles. These experiments suggest that vesicles assembled in dilute, mixed solutions are depleted of the shorter-chain-length lipid species, a finding that carries implications for the composition of primitive cell membranes., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Structural studies of modified nucleobases in RNA duplexes are critical for developing a full understanding of the stability and specificity of RNA base pairing. 2-Thio-uridine (s(2)U) is a modified nucleobase found in certain tRNAs. Thermodynamic studies have evaluated the effects of s(2)U on base pairing in RNA, where it has been shown to stabilize U:A pairs and destabilize U:G wobble pairs. Surprisingly, no high-resolution crystal structures of s(2)U-containing RNA duplexes have yet been reported. We present here two high-resolution crystal structures of heptamer RNA duplexes (5'-uagcs(2)Ucc-3' paired with 3'-aucgAgg-5' and with 3'-aucgUgg-5') containing s(2)U:A and s(2)U:U pairs, respectively. For comparison, we also present the structures of their native counterparts solved under identical conditions. We found that replacing O2 with S2 stabilizes the U:A base pair without any detectable structural perturbation. In contrast, an s(2)U:U base pair is strongly stabilized in one specific U:U pairing conformation out of four observed for the native U:U base pair. This s(2)U:U stabilization appears to be due at least in part to an unexpected sulfur-mediated hydrogen bond. This work provides additional insights into the effects of 2-thio-uridine on RNA base pairing.

We describe a new discovery technology that uses mRNA-display to rapidly synthesize and screen macrocyclic peptide libraries to explore a valuable region of chemical space typified by natural products. This technology allows high-affinity peptidic macrocycles containing modified backbones and unnatural side chains to be readily selected based on target binding. Success stories covering the first examples of these libraries suggest that they could be used for the discovery of intracellular protein-protein interaction inhibitors, highly selective enzyme inhibitors or synthetic replacements for monoclonal antibodies. The review concludes with a look to the future regarding how this technology might be improved with respect to library design for cell permeability and bioavailability., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The mixture of 2'-5' and 3'-5' linkages generated during the nonenzymatic replication of RNA has long been regarded as a central problem for the origin of the RNA world. However, we recently observed that both a ribozyme and an RNA aptamer retain considerable functionality in the presence of prebiotically plausible levels of linkage heterogeneity. To better understand the RNA structure and function in the presence of backbone linkage heterogeneity, we obtained high-resolution X-ray crystal structures of a native 10-mer RNA duplex (1.32 Å) and two variants: one containing one 2'-5' linkage per strand (1.55 Å) and one containing three such linkages per strand (1.20 Å). We found that RNA duplexes adjust their local structures to accommodate the perturbation caused by 2'-5' linkages, with the flanking nucleotides buffering the disruptive effects of the isomeric linkage and resulting in a minimally altered global structure. Although most 2'-linked sugars were in the expected 2'-endo conformation, some were partially or fully in the 3'-endo conformation, suggesting that the energy difference between these conformations was relatively small. Our structural and molecular dynamic studies also provide insight into the diminished thermal and chemical stability of the duplex state associated with the presence of 2'-5' linkages. Our results contribute to the view that a low level of 2'-5' substitution would not have been fatal in an early RNA world and may in contrast have been helpful for both the emergence of nonenzymatic RNA replication and the early evolution of functional RNAs.

The five-membered furanose ring is a central component of the chemical structure of biological nucleic acids. The conformations of the furanose ring can be analytically described using the concept of pseudorotation, and for RNA and DNA they are dominated by the C2'-endo and C3'-endo conformers. While the free energy difference between these two conformers can be inferred from NMR measurements, a free energy landscape of the complete pseudorotation cycle of nucleic acids in solution has remained elusive. Here, we describe a new free energy calculation method for molecular dynamics (MD) simulations using the two pseudorotation parameters directly as the collective variables. To validate our approach, we calculated the free energy surface of ribose pseudorotation in guanosine and 2'-deoxyguanosine. The calculated free energy landscape reveals not only the relative stability of the different pseudorotation conformers, but also the main transition path between the stable conformations. Applying this method to a standard A-form RNA duplex uncovered the expected minimum at the C3'-endo state. However, at a 2'-5' linkage, the minimum shifts to the C2'-endo conformation. The free energy of the C3'-endo conformation is 3 kcal/mol higher due to a weaker hydrogen bond and a reduced base stacking interaction. Unrestrained MD simulations suggest that the conversion from C3'-endo to C2'-endo and vice versa is on the nanosecond and microsecond time scale, respectively. These calculations suggest that 2'-5' linkages may enable folded RNAs to sample a wider spectrum of their pseudorotation conformations.

Threose nucleic acid (TNA) is a potential alternative genetic material that may have played a role in the early evolution of life. We have developed a novel synthesis of 2'-amino modified TNA nucleosides (2'-NH2-TNA) based on a cycloaddition reaction between a glycal and an azodicarboxylate, followed by direct nucleosidation of the cycloadduct. Using this route, we synthesized the thymine and guanine 2'-NH2-TNA nucleosides in seven steps with 24% and 12% overall yield, respectively. We then phosphorylated the guanine nucleoside on the 3'-hydroxyl, activated the phosphate as the 2-methylimidazolide, and tested the ability of the activated nucleotide to copy C4 RNA, DNA, and TNA templates by nonenzymatic primer extension. We measured pseudo-first-order rate constants for the first nucleotide addition step of 1.5, 0.97, and 0.57 h(-1) on RNA, DNA, and TNA templates, respectively, at pH 7.5 and 4 °C with 150 mM NaCl, 100 mM N-(hydroxylethyl)imidazole catalyst, and 5 mM activated nucleotide. The activated nucleotide hydrolyzed with a rate constant of 0.39 h(-1), causing the polymerization reaction to stall before complete template copying could be achieved. These extension rates are more than 1 order of magnitude slower than those for amino-sugar ribonucleotides under the same conditions, and copying of the TNA template, which best represented a true self-copying reaction, was the slowest of all. The poor kinetics of 2'-NH2-TNA template copying could give insight into why TNA was ultimately not used as a genetic material by biological systems.

Compartmentalization in a prebiotic setting is an important aspect of early cell formation and is crucial for the development of an artificial protocell system that effectively couples genotype and phenotype. Aqueous two-phase systems (ATPSs) and complex coacervates are phase separation phenomena that lead to the selective partitioning of biomolecules and have recently been proposed as membrane-free protocell models. We show in this study through fluorescence recovery after photobleaching (FRAP) microscopy that despite the ability of such systems to effectively concentrate RNA, there is a high rate of RNA exchange between phases in dextran/polyethylene glycol ATPS and ATP/poly-L-lysine coacervate droplets. In contrast to fatty acid vesicles, these systems would not allow effective segregation and consequent evolution of RNA, thus rendering these systems ineffective as model protocells.

The complexity of even the simplest known life forms makes efforts to synthesize living cells from inanimate components seem like a daunting task. However, recent progress toward the creation of synthetic cells, ranging from simple protocells to artificial cells approaching the complexity of bacteria, suggests that the synthesis of life is now a realistic goal. Protocell research, fueled by advances in the biophysics of primitive membranes and the chemistry of nucleic acid replication, is providing new insights into the origin of cellular life. Parallel efforts to construct more complex artificial cells, incorporating translational machinery and protein enzymes, are providing information about the requirements for protein-based life. We discuss recent advances and remaining challenges in the synthesis of artificial cells, the possibility of creating new forms of life distinct from existing biology, and the promise of this research for gaining a deeper understanding of the nature of living systems.

Efforts to recreate a prebiotically plausible protocell, in which RNA replication occurs within a fatty acid vesicle, have been stalled by the destabilizing effect of Mg(2+) on fatty acid membranes. Here we report that the presence of citrate protects fatty acid membranes from the disruptive effects of high Mg(2+) ion concentrations while allowing RNA copying to proceed, while also protecting single-stranded RNA from Mg(2+)-catalyzed degradation. This combination of properties has allowed us to demonstrate the chemical copying of RNA templates inside fatty acid vesicles, which in turn allows for an increase in copying efficiency by bathing the vesicles in a continuously refreshed solution of activated nucleotides.

Recent advances suggest that it may be possible to construct simple artificial cells from two subsystems: a self-replicating cell membrane and a self-replicating genetic polymer. Although multiple pathways for the growth and division of model protocell membranes have been characterized, no self-replicating genetic material is yet available. Nonenzymatic template-directed synthesis of RNA with activated ribonucleotide monomers has led to the copying of short RNA templates; however, these reactions are generally slow (taking days to weeks) and highly error prone. N3'-P5'-linked phosphoramidate DNA (3'-NP-DNA) is similar to RNA in its overall duplex structure, and is attractive as an alternative to RNA because the high reactivity of its corresponding monomers allows rapid and efficient copying of all four nucleobases on homopolymeric RNA and DNA templates. Here we show that both homopolymeric and mixed-sequence 3'-NP-DNA templates can be copied into complementary 3'-NP-DNA sequences. G:T and A:C wobble pairing leads to a high error rate, but the modified nucleoside 2-thiothymidine suppresses wobble pairing. We show that the 2-thiothymidine modification increases both polymerization rate and fidelity in the copying of a 3'-NP-DNA template into a complementary strand of 3'-NP-DNA. Our results suggest that 3'-NP-DNA has the potential to serve as the genetic material of artificial biological systems.

The advent of Darwinian evolution required the emergence of molecular mechanisms for the heritable variation of fitness. One model for such a system involves competing protocell populations, each consisting of a replicating genetic polymer within a replicating vesicle. In this model, each genetic polymer imparts a selective advantage to its protocell by, for example, coding for a catalyst that generates a useful metabolite. Here, we report a partial model of such nascent evolutionary traits in a system that consists of fatty-acid vesicles containing a dipeptide catalyst, which catalyses the formation of a second dipeptide. The newly formed dipeptide binds to vesicle membranes, which imparts enhanced affinity for fatty acids and thus promotes vesicle growth. The catalysed dipeptide synthesis proceeds with higher efficiency in vesicles than in free solution, which further enhances fitness. Our observations suggest that, in a replicating protocell with an RNA genome, ribozyme-catalysed peptide synthesis might have been sufficient to initiate Darwinian evolution.

A plausible process for non-enzymatic RNA replication would greatly simplify models of the transition from prebiotic chemistry to simple biology. However, all known conditions for the chemical copying of an RNA template result in the synthesis of a complementary strand that contains a mixture of 2'-5' and 3'-5' linkages, rather than the selective synthesis of only 3'-5' linkages as found in contemporary RNA. Here we show that such backbone heterogeneity is compatible with RNA folding into defined three-dimensional structures that retain molecular recognition and catalytic properties and, therefore, would not prevent the evolution of functional RNAs such as ribozymes. Moreover, the same backbone heterogeneity lowers the melting temperature of RNA duplexes that would otherwise be too stable for thermal strand separation. By allowing copied strands to dissociate, this heterogeneity may have been one of the essential features that allowed RNA to emerge as the first biopolymer.

A fast and accurate pathway for nonenzymatic RNA replication would simplify models for the emergence of the RNA world from the prebiotic chemistry of the early earth. However, numerous difficulties stand in the way of an experimental demonstration of effective nonenzymatic RNA replication. To gain insight into the necessary properties of potentially self-replicating informational polymers, we have studied several model systems based on amino-sugar nucleotides. Here we describe the synthesis of N3'-P5'-linked phosphoramidate DNA (3'-NP-DNA) by the template-directed polymerization of activated 3'-amino-2',3'-dideoxyribonucleotides. 3'-NP-DNA is an interesting model because of its very RNA-like A-type duplex conformation and because activated 3'-amino-2',3'-dideoxyribonucleotides are much more reactive than the corresponding activated ribonucleotides. In contrast to our previous studies with 2'-amino-2',3'-dideoxyribonucleotides (for which G and C but not A and T exhibit efficient template copying), we have found that all four canonical 3'-amino-2',3'-dideoxyribonucleotides (G, C, A, and T) polymerize efficiently on RNA templates. RNA templates are generally superior to DNA templates, and oligo-ribo-T templates are superior to oligo-ribo-U templates, which are the least efficient of the RNA homopolymer templates. We have also found that activation of 3'-aminonucleotides with 2-methylimidazole results in a ca. 10-fold higher polymerization rate relative to activation with imidazole, an observation that parallels earlier findings with ribonucleotides. We discuss the implications of our experiments for the possibility of self-replication in the 3'-NP-DNA and RNA systems.

Here we describe a method for preparing fatty acid micelles. The method for adding micelles to a buffered solution containing fatty acid or phospholipid vesicles is also discussed., (© 2013 Elsevier Inc. All rights reserved.)

The vesicle extrusion method is used for preparing small (e.g., 100 nm diameter) monodisperse (uniform-sized) vesicles. Polycarbonate track-etched membranes with pores generally ranging from 50 to 200 nm in diameter and a hand-held mini-extruder are used for such a procedure. Extrusion through small (100 nm in diameter or smaller) pores also ensures that the vesicle population is predominantly unilamellar., (© 2013 Elsevier Inc. All rights reserved.)

Prepare polydisperse, multilamellar vesicles by rehydrating a thin film of fatty acids or phospholipids., (© 2013 Elsevier Inc. All rights reserved.)

The first protocell membranes may have assembled from fatty acids and related single-chain lipids available in the prebiotic environment. Prior to the evolution of complex cellular machinery, spontaneous protocell membrane growth and division had to result from the intrinsic physicochemical properties of these molecules, in the context of specific environmental conditions. Depending on the nature of the chemical and physical environment, fatty acids can partition between several different phases, including soluble monomers, micelles, and lamellar vesicles. Here we address the concentration dependence of fatty acid aggregation, which is dominated by entropic considerations. We quantitatively distinguish between fatty acid phases using a combination of physical and spectroscopic techniques, including the use of the fluorescent fatty acid analogue Laurdan, whose emission spectrum is sensitive to structural differences between micellar and lamellar aggregates. We find that the monomer-aggregate transition largely follows a characteristic pseudophase model of molecular aggregation but that the composition of the aggregate phase is also concentration dependent. At low amphiphile concentrations above the critical aggregate concentration, vesicles coexist with a significant proportion of micelles, while more concentrated solutions favor the lamellar vesicle phase. We subsequently show that the micelle-vesicle equilibrium can be used to drive the growth of pre-existing vesicles upon an increase in amphiphile concentration either through solvent evaporation or following the addition of excess lipids. We propose a simple model for a primitive environmentally driven cell cycle, in which protocell membrane growth results from evaporative concentration, followed by shear force or photochemically induced division.

We propose a novel pathway for the prebiotic synthesis of 2'-deoxynucleotides. Consideration of the constitutional chemical relationships between glycolaldehyde and β-mercapto-acetaldehyde, and the corresponding proteinogenic amino acids, serine and cysteine, led us to explore the consequences of the corresponding sulfur substitution for our previously proposed pathways leading to the canonical ribonucleotides. We demonstrate that just as 2-aminooxazole-an important prebiotic ribonucleotide precursor-is readily formed from glycolaldehyde and cyanamide, so is 2-aminothiazole formed from β-mercapto-acetaldehyde and cyanamide in water at neutral pH. Indeed, both the oxazole and the thiazole can be formed together in a one-pot reaction, and can be co-purified by crystallization or sublimation. We then show that 2-aminothiazole can take part in a 3-component carbon-carbon bond-forming reaction in water that leads to the diastereoselective synthesis of masked 2'-thiosugars regiospecifically tethered to purine precursors, which would lead to 2'-deoxynucleotides upon desulfurization. The possibility of an abiotic route to the 2'-deoxynucleotides provides a new perspective on the evolutionary origins of DNA. We also show that 2-aminothiazole is able to sequester, through reversible aminal formation, the important nucleotide precursors glycolaldehyde and glyceraldehyde in a stable, crystalline form.

There is a great demand for the discovery of new therapeutic molecules that combine the high specificity and affinity of biologic drugs with the bioavailability and lower cost of small molecules. Small, natural-product-like peptides hold great promise in bridging this gap; however, access to libraries of these compounds has been a limitation. Since ribosomal peptides may be subjected to in vitro selection techniques, the generation of extremely large libraries (>10(13)) of highly modified macrocyclic peptides may provide a powerful alternative for the generation and selection of new useful bioactive molecules. Moreover, the incorporation of many non-proteinogenic amino acids into ribosomal peptides in conjunction with macrocyclization should enhance the drug-like features of these libraries. Here we show that mRNA-display, a technique that allows the in vitro selection of peptides, can be applied to the evolution of macrocyclic peptides that contain a majority of unnatural amino acids. We describe the isolation and characterization of two such unnatural cyclic peptides that bind the protease thrombin with low nanomolar affinity, and we show that the unnatural residues in these peptides are essential for the observed high-affinity binding. We demonstrate that the selected peptides are tight-binding inhibitors of thrombin, with K(i)(app) values in the low nanomolar range. The ability to evolve highly modified macrocyclic peptides in the laboratory is the first crucial step toward the facile generation of useful molecular reagents and therapeutic lead molecules that combine the advantageous features of biologics with those of small-molecule drugs.

Prior to the evolution of complex biochemical machinery, the growth and division of simple primitive cells (protocells) must have been driven by environmental factors. We have previously demonstrated two pathways for fatty acid vesicle growth in which initially spherical vesicles grow into long filamentous vesicles; division is then mediated by fluid shear forces. Here we describe a different pathway for division that is independent of external mechanical forces. We show that the illumination of filamentous fatty acid vesicles containing either a fluorescent dye in the encapsulated aqueous phase, or hydroxypyrene in the membrane, rapidly induces pearling and subsequent division in the presence of thiols. The mechanism of this photochemically driven pathway most likely involves the generation of reactive oxygen species, which oxidize thiols to disulfide-containing compounds that associate with fatty acid membranes, inducing a change in surface tension and causing pearling and subsequent division. This vesicle division pathway provides an alternative route for the emergence of early self-replicating cell-like structures, particularly in thiol-rich surface environments where UV-absorbing polycyclic aromatic hydrocarbons (PAHs) could have facilitated protocell division. The subsequent evolution of cellular metabolic processes controlling the thiol:disulfide redox state would have enabled autonomous cellular control of the timing of cell division, a major step in the origin of cellular life.

In this report we present a method to identify functional artificial lantipeptides. In vitro translation coupled with an enzyme-free protocol for posttranslational modification allows preparation of more than 10(11) different lanthionine containing peptides. This diversity can be searched for functional molecules using mRNA-lantipeptide display. We validated this approach by isolating binders toward Sortase A, a transamidase which is required for virulence of Staphylococcus aureus. The interaction of selected lantipeptides with Sortase A is highly dependent on the presence of a (2S,6R)-lanthionine in the peptide and an active conformation of the protein.

Template-directed polymerization of chemically activated ribonucleotide monomers, such as nucleotide 5'-phosphorimidazolides, has been studied as a model for nonenzymatic RNA replication during the origin of life. Kinetic studies of the polymerization of various nucleotide monomers on oligonucleotide templates have suggested that the A-form (C3'-endo sugar pucker) conformation is optimal for both monomers and templates for efficient copying. However, RNA monomers are predominantly in the C2'-endo conformation when free in solution, except for cytidine, which is approximately equally distributed between the C2'-endo and C3'-endo conformations. We hypothesized that ribonucleotides undergo a switch in sugar pucker upon binding to an A-type template and that this conformational switch allows or enhances subsequent polymerization. We used transferred nuclear Overhauser effect spectroscopy (TrNOESY), which can be used for specific detection of the bound conformation of small-molecule ligands with relatively weak affinity to receptors, to study the interactions between nucleotide 5'-phosphorimidazolides and single-stranded oligonucleotide templates. We found that the sugar pucker of activated ribonucleotides switches from C2'-endo in the free state to C3'-endo upon binding to an RNA template. This switch occurs only on RNA and not on DNA templates. Furthermore, activated 2'-deoxyribonucleotides maintain a C2'-endo sugar pucker in both the free and template-bound states. Our results provide a structural explanation for the observations that activated ribonucleotides are superior to activated deoxyribonucleotides and that RNA templates are superior to DNA templates in template-directed nonenzymatic primer-extension reactions., (© 2012 American Chemical Society)

The accumulation of pure, concentrated chemical building blocks, from which the essential components of protocells could be assembled, has long been viewed as a necessary, but extremely difficult step on the pathway to the origin of life. However, recent experiments have shown that moderately increasing the complexity of a set of chemical inputs can in some cases lead to a dramatic simplification of the resulting reaction products. Similarly, model protocell membranes composed of certain mixtures of amphiphilic molecules have superior physical properties than membranes composed of single amphiphiles. Moreover, membrane self-assembly under simple and natural conditions gives rise to heterogeneous mixtures of large multi-lamellar vesicles, which are predisposed to a robust pathway of growth and division that simpler and more homogeneous small unilamellar vesicles cannot undergo. Might a similar relaxation of the constraints on building block purity and homogeneity actually facilitate the difficult process of nucleic acid replication? Several arguments suggest that mixtures of monomers and short oligonucleotides may enable the chemical copying of polynucleotides of sufficient length and sequence complexity to allow for the emergence of the first nucleic acid catalysts. The question of the origin of life may become less daunting once the constraints of overly well-defined laboratory experiments are appropriately relaxed.

Multiple lines of evidence support the hypothesis that the early evolution of life was dominated by RNA, which can both transfer information from generation to generation through replication directed by base-pairing, and carry out biochemical activities by folding into functional structures. To understand how life emerged from prebiotic chemistry we must therefore explain the steps that led to the emergence of the RNA world, and in particular, the synthesis of RNA. The generation of pools of highly pure ribonucleotides on the early Earth seems unlikely, but the presence of alternative nucleotides would support the assembly of nucleic acid polymers containing nonheritable backbone heterogeneity. We suggest that homogeneous monomers might not have been necessary if populations of heterogeneous nucleic acid molecules could evolve reproducible function. For such evolution to be possible, function would have to be maintained despite the repeated scrambling of backbone chemistry from generation to generation. We have tested this possibility in a simplified model system, by using a T7 RNA polymerase variant capable of transcribing nucleic acids that contain an approximately 11 mixture of deoxy- and ribonucleotides. We readily isolated nucleotide-binding aptamers by utilizing an in vitro selection process that shuffles the order of deoxy- and ribonucleotides in each round. We describe two such RNA/DNA mosaic nucleic acid aptamers that specifically bind ATP and GTP, respectively. We conclude that nonheritable variations in nucleic acid backbone structure may not have posed an insurmountable barrier to the emergence of functionality in early nucleic acids.

We have devised a protocol for enzyme-free insertion of dehydroalanine, dehydrobutyrine and thioether crosslinks into translated peptides. In vitro translation using 4-selenalysine and 4-selenoisoleucine as substitutes for lysine and isoleucine yields peptides that can be converted to polycyclic structures using mild chemistry in water. This methodology presents a gateway for exploring the potential of artificial lantipeptides as scaffolds for drug development.

To understand the emergence of Darwinian evolution, it is necessary to identify physical mechanisms that enabled primitive cells to compete with one another. Whereas all modern cell membranes are composed primarily of diacyl or dialkyl glycerol phospholipids, the first cell membranes are thought to have self-assembled from simple, single-chain lipids synthesized in the environment. We asked what selective advantage could have driven the transition from primitive to modern membranes, especially during early stages characterized by low levels of membrane phospholipid. Here we demonstrate that surprisingly low levels of phospholipids can drive protocell membrane growth during competition for single-chain lipids. Growth results from the decreasing fatty acid efflux from membranes with increasing phospholipid content. The ability to synthesize phospholipids from single-chain substrates would have therefore been highly advantageous for early cells competing for a limited supply of lipids. We show that the resulting increase in membrane phospholipid content would have led to a cascade of new selective pressures for the evolution of metabolic and transport machinery to overcome the reduced membrane permeability of diacyl lipid membranes. The evolution of phospholipid membranes could thus have been a deterministic outcome of intrinsic physical processes and a key driving force for early cellular evolution.

The recent development of a sequential, high-yielding route to activated pyrimidine nucleotides, under conditions thought to be prebiotic, is an encouraging step toward the greater goal of a plausible prebiotic pathway to RNA and the potential for an RNA world. However, this synthesis has led to a disparity in the methodology available for stepwise construction of the canonical pyrimidine and purine nucleotides. To address this problem, and further explore prebiotically accessible chemical systems, we have developed a high-yielding, aqueous, one-pot, multicomponent reaction that tethers masked-sugar moieties to prebiotically plausible purine precursors. A pH-dependent three-component reaction system has been discovered that utilizes key nucleotide synthons 2-aminooxazole and 5-aminoimidazoles, which allows the first divergent purine/pyrimidine synthesis to be proposed. Due to regiospecific aminoimidazole tethering, the pathway allows N9 purination only, thus suggesting the first prebiotically plausible mechanism for regiospecific N9 purination.

Understanding the origin of cellular life on Earth requires the discovery of plausible pathways for the transition from complex prebiotic chemistry to simple biology, defined as the emergence of chemical assemblies capable of Darwinian evolution. We have proposed that a simple primitive cell, or protocell, would consist of two key components: a protocell membrane that defines a spatially localized compartment, and an informational polymer that allows for the replication and inheritance of functional information. Recent studies of vesicles composed of fatty-acid membranes have shed considerable light on pathways for protocell growth and division, as well as means by which protocells could take up nutrients from their environment. Additional work with genetic polymers has provided insight into the potential for chemical genome replication and compatibility with membrane encapsulation. The integration of a dynamic fatty-acid compartment with robust, generalized genetic polymer replication would yield a laboratory model of a protocell with the potential for classical Darwinian biological evolution, and may help to evaluate potential pathways for the emergence of life on the early Earth. Here we discuss efforts to devise such an integrated protocell model.

We describe an approach for performing single-molecule binding experiments on time scales from hours to days, allowing for the observation of slower kinetics than have been previously investigated by single-molecule techniques. Total internal reflection fluorescence microscopy is used to image the binding of labeled ligand to molecules specifically coupled to the surface of an optically transparent flow cell. Long-duration experiments are enabled by ensuring sufficient positional, chemical, thermal, and image stability. Principal components of this experimental stability include illumination timing, solution replacement, and chemical treatment of solution to reduce photodamage and photobleaching; and autofocusing to correct for spatial drift.

The frequency of errors during genome replication limits the amount of functionally important information that can be passed on from generation to generation. During the origin of life, mutation rates are thought to have been quite high, raising a classic chicken-and-egg paradox: could nonenzymatic replication propagate sequences accurately enough to allow for the emergence of heritable function? Here we show that the theoretical limit on genomic information content may increase substantially as a consequence of dramatically slowed polymerization after mismatches. As a result of postmismatch stalling, accurate copies of a template tend to be completed more rapidly than mutant copies and the accurate copies can therefore begin a second round of replication more quickly. To quantify this effect, we characterized an experimental model of nonenzymatic, template-directed nucleic acid polymerization. We found that most mismatches decrease the rate of primer extension by more than 2 orders of magnitude relative to a matched (Watson-Crick) control. A chemical replication system with this property would be able to propagate sequences long enough to have function. Our study suggests that the emergence of functional sequences during the origin of life would be possible even in the face of the high intrinsic error rates of chemical replication.