Progress toward synthetic cells.

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.

Mettl1/Wdr4-Mediated m7G tRNA Methylome Is Required for Normal mRNA Translation and Embryonic Stem Cell Self-Renewal and Differentiation.

tRNAs are subject to numerous modifications, including methylation. Mutations in the human N7-methylguanosine (m7G) methyltransferase complex METTL1/WDR4 cause primordial dwarfism and brain malformation, yet the molecular and cellular function in mammals is not well understood. We developed m7G methylated tRNA immunoprecipitation sequencing (MeRIP-seq) and tRNA reduction and cleavage sequencing (TRAC-seq) to reveal the m7G 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 m7G tRNA methylome and highlights its essential role in ESCs with links to human disease.

Transient states during the annealing of mismatched and bulged oligonucleotides.

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

Constraints on the emergence of RNA through non-templated primer extension with mixtures of potentially prebiotic nucleotides.

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

Passive endocytosis in model protocells.

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

Enhanced nonenzymatic RNA copying with in-situ activation of short oligonucleotides.

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

The absence of a prebiotically plausible pathway for the efficient nonenzymatic copying of RNAs remains a major obstacle towards constructing self-replicating protocells that emulate early lifeforms. We demonstrate the activation of short oligonucleotides and the subsequent formation of monomer-bridged-oligonucleotide species, leading to efficient nonenzymatic template copying in the same reaction mixture. Our findings suggest that in-situ activated mixtures of mono- and oligo-nucleotides would significantly outperform mononucleotides in driving the copying of arbitrary RNA sequences. Our demonstration that multiple steps in the pathway from activation chemistry to RNA copying can occur together in a single complex environment simplifies this aspect of the origin of life.

Freeze-thaw cycles enable a prebiotically plausible and continuous pathway from nucleotide activation to nonenzymatic RNA copying.

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

Nonenzymatic assembly of active chimeric ribozymes from aminoacylated RNA oligonucleotides.

Aminoacylated transfer RNAs, which harbor a covalent linkage between amino acids and RNA, are a universally conserved feature of life. Because they are essential substrates for ribosomal translation, aminoacylated oligonucleotides must have been present in the RNA world prior to the evolution of the ribosome. One possibility we are exploring is that the aminoacyl ester linkage served another function before being recruited for ribosomal protein synthesis. The nonenzymatic assembly of ribozymes from short RNA oligomers under realistic conditions remains a key challenge in demonstrating a plausible pathway from prebiotic chemistry to the RNA world. Here, we show that aminoacylated RNAs can undergo template-directed assembly into chimeric amino acid-RNA polymers that are active ribozymes. We demonstrate that such chimeric polymers can retain the enzymatic function of their all-RNA counterparts by generating chimeric hammerhead, RNA ligase, and aminoacyl transferase ribozymes. Amino acids with diverse side chains form linkages that are well tolerated within the RNA backbone and, in the case of an aminoacyl transferase, even in its catalytic center, potentially bringing novel functionalities to ribozyme catalysis. Our work suggests that aminoacylation chemistry may have played a role in primordial ribozyme assembly. Increasing the efficiency of this process provides an evolutionary rationale for the emergence of sequence and amino acid-specific aminoacyl-RNA synthetase ribozymes, which could then have generated the substrates for ribosomal protein synthesis.

Simulations predict preferred Mg2+ coordination in a nonenzymatic primer-extension reaction center.

The mechanism by which genetic information was copied prior to the evolution of ribozymes is of great interest because of its importance to the origin of life. The most effective known process for the nonenzymatic copying of an RNA template is primer extension by a two-step pathway in which 2-aminoimidazole-activated nucleotides first react with each other to form an imidazolium-bridged intermediate that subsequently reacts with the primer. Reaction kinetics, structure-activity relationships, and X-ray crystallography have provided insight into the overall reaction mechanism, but many puzzles remain. In particular, high concentrations of Mg2+ are required for efficient primer extension, but the mechanism by which Mg2+ accelerates primer extension remains unknown. By analogy with the mechanism of DNA and RNA polymerases, a role for Mg2+ in facilitating the deprotonation of the primer 3'-hydroxyl is often assumed, but no catalytic metal ion is seen in crystal structures of the primer-extension complex. To explore the potential effects of Mg2+ binding in the reaction center, we performed atomistic molecular dynamics simulations of a series of modeled complexes in which a Mg2+ ion was placed in the reaction center with inner-sphere coordination with different sets of functional groups. Our simulations suggest that coordination of a Mg2+ ion with both O3' of the terminal primer nucleotide and the pro-Sp nonbridging oxygen of the reactive phosphate of an imidazolium-bridged dinucleotide would help to pre-organize the structure of the primer/template substrate complex to favor the primer-extension reaction. Our results suggest that the catalytic metal ion may play an important role in overcoming electrostatic repulsion between a deprotonated O3' and the reactive phosphate of the bridged dinucleotide and lead to testable predictions of the mode of Mg2+ binding that is most relevant to catalysis of primer extension.

RNA Complexes with Nicks and Gaps: Thermodynamic and Kinetic Effects of Coaxial Stacking and Dangling Ends.

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

Diaminopurine in Nonenzymatic RNA Template Copying.

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

Catalytic Metal Ion-Substrate Coordination during Nonenzymatic RNA Primer Extension.

Nonenzymatic template-directed RNA copying requires catalysis by divalent metal ions. The primer extension reaction involves the attack of the primer 3'-hydroxyl on the adjacent phosphate of a 5'-5'-imidazolium-bridged dinucleotide substrate. However, the nature of the interaction of the catalytic metal ion with the reaction center remains unclear. To explore the coordination of the catalytic metal ion with the imidazolium-bridged dinucleotide substrate, we examined catalysis by oxophilic and thiophilic metal ions with both diastereomers of phosphorothioate-modified substrates. We show that Mg2+ and Cd2+ exhibit opposite preferences for the two phosphorothioate substrate diastereomers, indicating a stereospecific interaction of the divalent cation with one of the nonbridging phosphorus substituents. High-resolution X-ray crystal structures of the products of primer extension with phosphorothioate substrates reveal the absolute stereochemistry of this interaction and indicate that catalysis by Mg2+ involves inner-sphere coordination with the nonbridging phosphate oxygen in the pro-SP position, while thiophilic cadmium ions interact with sulfur in the same position, as in one of the two phosphorothioate substrates. These results collectively suggest that during nonenzymatic RNA primer extension with a 5'-5'-imidazolium-bridged dinucleotide substrate the interaction of the catalytic Mg2+ ion with the pro-SP oxygen of the reactive phosphate plays a crucial role in the metal-catalyzed SN2(P) reaction.

Unusual Base Pair between Two 2-Thiouridines and Its Implication for Nonenzymatic RNA Copying.

2-Thiouridine (s2U) is a nucleobase modification that confers enhanced efficiency and fidelity both on modern tRNA codon translation and on nonenzymatic and ribozyme-catalyzed RNA copying. We have discovered an unusual base pair between two 2-thiouridines that stabilizes an RNA duplex to a degree that is comparable to that of a native A:U base pair. High-resolution crystal structures indicate similar base-pairing geometry and stacking interactions in duplexes containing s2U:s2U compared to those with U:U pairs. Notably, the C═O···H-N hydrogen bond in the U:U pair is replaced with a C═S···H-N hydrogen bond in the s2U:s2U base pair. The thermodynamic stability of the s2U:s2U base pair suggested that this self-pairing might lead to an increased error frequency during nonenzymatic RNA copying. However, competition experiments show that s2U:s2U base-pairing induces only a low level of misincorporation during nonenzymatic RNA template copying because the correct A:s2U base pair outcompetes the slightly weaker s2U:s2U base pair. In addition, even if an s2U is incorrectly incorporated, the addition of the next base is greatly hindered. This strong stalling effect would further increase the effective fidelity of nonenzymatic RNA copying with s2U. Our findings suggest that s2U may enhance the rate and extent of nonenzymatic copying with only a minimal cost in fidelity.

Hybridization kinetics of out-of-equilibrium mixtures of short RNA oligonucleotides.

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

Kinetic explanations for the sequence biases observed in the nonenzymatic copying of RNA templates.

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

Thermodynamics and kinetics of DNA and RNA dinucleotide hybridization to gaps and overhangs.

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

Small-Molecule Organocatalysis Facilitates In Situ Nucleotide Activation and RNA Copying.

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

Experimental Tests of the Virtual Circular Genome Model for Nonenzymatic RNA Replication.

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

The virtual circular genome model for primordial RNA replication.

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

RNA-Catalyzed RNA Ligation within Prebiotically Plausible Model Protocells.

Demonstrating RNA catalysis within prebiotically relevant models of primordial cells (protocells) remains a challenge in origins of life research. Fatty acid vesicles encapsulating genomic and catalytic RNAs (ribozymes) are attractive models for protocells; however, RNA catalysis has largely been incompatible with fatty acid vesicles due to their instability in the presence of Mg2+ at the concentrations required for ribozyme function. Here, we report a ribozyme that catalyzes template-directed RNA ligation at low Mg2+ concentrations and thus remains active within stable vesicles. Ribose and adenine, both prebiotically relevant molecules, were found to greatly reduce Mg2+ -induced RNA leakage from vesicles. When we co-encapsulated the ribozyme, substrate, and template within fatty acid vesicles, we observed efficient RNA-catalyzed RNA ligation upon subsequent addition of Mg2+ . Our work shows that RNA-catalyzed RNA assembly can occur efficiently within prebiotically plausible fatty acid vesicles and represents a step toward the replication of primordial genomes within self-replicating protocells.