Biology subjects

Szostak, J. W.

Publications and source records attributed to Szostak, J. W..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

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, due to their higher intrinsic reactivity and their higher template affinity. 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 findings pave the way for future research into the activation of complex mixtures of mono- and oligonucleotides for the enhanced copying and potentially the replication of arbitrary RNA sequences.

biochemistry↗

bioRxiv2023

Direct monitoring of the thermodynamics and kinetics of DNA and RNA dinucleotide dehybridization from gaps and overhangs

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

biophysics↗

bioRxiv2023

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 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.

biochemistry↗

bioRxiv2023

RNA conformation and metal ion coordination in the 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 to different sets of functional groups. Our simulations suggest that coordination of a Mg2+ ion to both O3' of the terminal primer nucleotide and the pro-Sp non- bridging oxygen of the reactive phosphate of an imidazolium-bridged dinucleotide would help to preorganize 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. Our simulations lead to testable predictions of the mode of Mg2+ binding that is most relevant to catalysis of primer extension. STATEMENT OF SIGNIFICANCEPrior to the evolution of complex enzymes, the replication of genetic material must have relied on nonenzymatic mechanisms. Nonenzymatic RNA template copying can be achieved through the extension of a primer by reaction with a 2-aminoimidazole (2AI) bridged dinucleotide in the presence of Mg2+. Despite progress in understanding the mechanism of this reaction, the catalytic role of Mg2+ remains poorly understood. Here, we present a series of molecular dynamics simulations of a model RNA primer-extension complex in different potential reactive conformations. We find that one configuration of both the 2AI moiety and coordination state of the Mg2+ promotes a geometry that is most favorable to reaction, suggesting a potential structural role for Mg2+ and providing insights to guide future experiments.

biophysics↗

bioRxiv2023

Experimental Tests of the Virtual Circular Genome Model for Non-enzymatic 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 extension on a subset of kinetically trapped partially base-paired configurations, followed by 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 length 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 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 pre-activated. We discuss the necessity of ongoing in-situ activation chemistry for continuous and accurate VCG replication.

biochemistry↗

bioRxiv2023

Passive endocytosis in protocells enables nutrient transport bypassing permeability limits

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.

biophysics↗

bioRxiv2022

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) as simple functions of oligonucleotide length, CG content, {Delta}G of hybridization and {Delta}G 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.

biochemistry↗

bioRxiv2021

Nonenzymatic loop-closing ligation generates RNA hairpins and is a template-free way to assemble functional RNAs

RNA hairpin loops are the predominant element of secondary structure in functional RNAs. The emergence of primordial functional RNAs, such as ribozymes that fold into complex structures that contain multiple hairpin loops, is generally thought to have been supported by template-directed ligation. However, template inhibition and RNA misfolding problems impede the emergence of function. Here we demonstrate that RNA hairpin loops can be synthesized directly from short RNA duplexes with single-stranded overhangs by nonenzymatic loop-closing ligation chemistry. We show that loop-closing ligation allows full-length functional ribozymes containing a hairpin loop to be assembled free of inhibitory template strands. This approach to the assembly of structurally complex RNAs suggests a plausible pathway for the emergence of functional RNAs before a full-length RNA copying process became available.

synthetic biology↗

bioRxiv2021

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. 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.

biochemistry↗

bioRxiv2021

Non-enzymatic assembly of active chimeric ribozymes from aminoacylated RNA oligonucleotides

Aminoacylated tRNAs, 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, 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. Significance StatementThe emergence of the primordial ribosome from the RNA World would have required access to aminoacylated RNA substrates. The spontaneous generation of such substrates without enzymes is inefficient, and it remains unclear how they could be selected for in a prebiotic milieu. In our study we identify a role for aminoacylated RNA in ribozyme assembly, a longstanding problem in the origin of life research. We show that aminoacylated RNAs, but not unmodified RNAs, rapidly assemble into chimeric amino acid-bridged ribozymes that retain their native enzymatic activity. Our work therefore addresses two key challenges within the origin-of-life field: we demonstrate assembly of functional ribozymes and we identify a potential evolutionary benefit for RNA aminoacylation that is independent of coded peptide translation.

biochemistry↗

bioRxiv2021

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 replication. Here we explore three sequential steps from this overall pathway: nucleotide activation, synthesis of imidazolium-bridged dinucleotides, and template-directed primer extension. We find that all three steps can take place in one reaction mixture, under conditions of multiple freeze-thaw cycles. Recent experiments have demonstrated a potentially prebiotic methyl isocyanide-based nucleotide activation chemistry. Unfortunately, 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 needed for primer extension. 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 primer extension 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. Significance StatementThe replication of RNA without the aid of evolved enzymes may have enabled the inheritance of useful molecular functions during the origin of life. Several key steps on the path to RNA replication have been studied in isolation, including chemical nucleotide activation, synthesis of a key reactive intermediate, and nonenzymatic RNA copying. Here we report a prebiotically plausible scenario under which these reactions can happen together under mutually compatible conditions. Thus, this pathway could potentially have operated in nature without the complicating requirement for exchange of materials between distinct environments.

biochemistry↗

bioRxiv2021

Competition between bridged dinucleotides and activated mononucleotides determines the error frequency of nonenzymatic RNA primer extension

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

molecular biology↗

bioRxiv2020

A Potential Role for Aminoacylation in Primordial RNA Copying Chemistry

Aminoacylated tRNAs are the substrates for ribosomal protein synthesis in all branches of life, implying an ancient origin for aminoacylation chemistry. In the 1970s, Orgel and colleagues reported potentially prebiotic routes to aminoacylated nucleotides and their RNA templated condensation to form amino acid bridged dinucleotides. However, it is unclear whether such reactions would have aided or impeded nonenzymatic RNA replication. Determining whether aminoacylated RNAs could have been advantageous in evolution prior to the emergence of protein synthesis remains a key challenge. We therefore tested the ability of aminoacylated RNA to participate in both templated primer extension and ligation reactions. We find that at low magnesium concentrations that favor fatty acid-based protocells, these reactions proceed orders of magnitude more rapidly than when initiated from the cis-diol of unmodified RNA. We further demonstrate that amino acid bridged RNAs can act as templates in a subsequent round of copying. Our results suggest that aminoacylation facilitated nonenzymatic RNA replication, thus outlining a potentially primordial functional link between aminoacylation chemistry and RNA replication. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/413815v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1e5f29dorg.highwire.dtl.DTLVardef@19b2a99org.highwire.dtl.DTLVardef@9aad0org.highwire.dtl.DTLVardef@ad47f7_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2020

Structural interpretation of the effects of threo-nucleotides on nonenzymatic template-directed polymerization

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

biochemistry↗

bioRxiv2020

Prebiotically Plausible Activation Chemistry Compatible with Non-enzymatic RNA Copying

The nonenzymatic replication of ribonucleic acid (RNA) oligonucleotides 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 remains a challenge. Here we report a novel pathway that enables the activation of RNA nucleotides in a manner that is compatible with template-directed nonenzymatic polymerization. We show that this pathway selectively yields the reactive imidazolium-bridged dinucleotide intermediate required for nonenzymatic template-directed RNA copying. Our results will enable more realistic prebiotic chemical simulations of RNA copying based on continuous in situ nucleotide activation.

biochemistry↗

bioRxiv2020

In search of the RNA world on Mars

Advances in origins of life research and prebiotic chemistry suggest that life as we know it may have emerged from an earlier RNA World. However, it has been difficult to reconcile the conditions used in laboratory experiments with real-world geochemical environments that may have existed on the early Earth and hosted the origin(s) of life. This challenge is in part due to geologic resurfacing and recycling that have erased the overwhelming majority of the Earths prebiotic history. We therefore propose that Mars, a planet frozen in time, comprised of many surfaces that have remained relatively unchanged since their formation >4 Gya, is the best alternative to search for environments consistent with geochemical requirements imposed by the RNA world. In this study we synthesize in situ and orbital observations of Mars and modeling of its early atmosphere into solutions containing a range of pHs and concentrations of prebiotically relevant metals (Fe2+, Mg2+, and Mn2+), spanning various candidate aqueous environments. We then experimentally determine RNA degradation kinetics due to metal-catalyzed hydrolysis and evaluate whether early Mars could have been permissive towards the accumulation of long-lived RNA polymers. Our results indicate that a Mg2+-rich basalt sourcing metals to a slightly acidic (pH 5.4) aqueous environment mediates the slowest rates of metal-catalyzed RNA hydrolysis, though geologic evidence and modeling of basalt weathering suggest that aquifers on Mars would be near neutral (pH [~]7). Moreover, oxidizing conditions on Mars have major consequences regarding the availability oxygen-sensitive prebiotic metals (i.e., Fe2+ and Mn2+) very early in its history due to increased RNA degradation rates and precipitation. Overall, 1) low pH better preserves RNA than basic conditions at high concentrations; 2) acidic to neutral pH environments with Fe2+ or Mn2+ will hydrolyze more RNA; and 3) alkaline environments with Mg2+ dramatically hydrolyze more RNA.

molecular biology↗

bioRxiv2020

Deep sequencing of nonenzymatic RNA primer extension

Life emerging in an RNA world is expected to propagate RNA as hereditary information, requiring some form of primitive replication without enzymes. Nonenzymatic template-directed RNA primer extension is a model of the polymerisation 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 sorts and analyses raw 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.

synthetic biology↗

bioRxiv2020

Bacterial carotenoids suppress Caenorhabditis elegans surveillance and defense of translational dysfunction

Microbial toxins and virulence factors often target the eukaryotic translation machinery. Caenorhabditis elegans surveils for such microbial attacks by monitoring translational competence, and if a deficit is detected, particular drug detoxification and bacterial defense genes are induced. The bacteria Kocuria rhizophila has evolved countermeasures to animal translational surveillance and defense pathways. Here, we used comprehensive genetic analysis of Kocuria rhizophila to identify the bacterial genetic pathways that inhibit C. elegans translational toxin surveillance and defense. Kocuria rhizophila mutations that disrupt its ability to disable animal immunity and defense map to multiple steps in the biosynthesis of a 50-carbon bacterial carotenoid from 5 carbon precursors. Extracts of the C50 carotenoid from wild type K. rhizophila could restore this bacterial anti-immunity activity to K. rhizophila carotenoid biosynthetic mutant. Corynebacterium glutamicum, also inhibits the C. elegans translation detoxification response by producing the C50 carotenoid decaprenoxanthin, and C. glutamicum carotenoid mutants are defective in this suppression of C. elegans detoxification. Consistent with the salience of these bacterial countermeasures to animal drug responses, bacterial carotenoids sensitize C. elegans to drugs that target translation and inhibit food aversion behaviors normally induced by protein translation toxins or mutations. The surveillance and response to toxins is mediated by signaling pathways conserved across animal phylogeny, suggesting that these bacterial carotenoids may also suppress such human immune and toxin responses.

genomics↗