Biology subjects

Szostak, J. W.

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

At least 19 recordsLinked to original sources

bioRxiv2026

tinyRNA-seq: An optimized approach to sequencing tiny RNAs and primitive RNA genomes

Very short RNAs play critical roles in modern biology, and are thought to have been crucial for genome replication during the origin of life. Next-generation sequencing is an essential tool for characterizing pools of small RNAs, but current library preparation methods suffer from strong size and sequence biases. Here we present tinyRNA-seq, an optimized library preparation method designed to minimize length- and sequence-dependent capture bias enabling the sequencing of RNA fragments as short as 2 nucleotides. We use degenerate adaptor regions to reduce ligation sequence bias and facilitate unique molecular identifier (UMI) installation. We benchmarked tinyRNA-seq against commercial kits using a model primordial RNA genome consisting of hundreds of defined oligonucleotides ranging from 2 to 12 nucleotides. tinyRNA-seq reproduced the input RNA distribution without the size and sequence bias of the commercial kits. tinyRNA-seq also enables the detection of de novo oligonucleotide generation, an important process for the origins of life. Applied to biologically derived small RNAs including miRNAs, piRNAs, and cityRNAs, tinyRNA-seq showed significantly lower capture bias and recovered a wider range of sequences than commercial kits. tinyRNA-seq may thus provide a more complete and quantitatively accurate representation of small RNAs from both biological and chemical sources. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=94 SRC="FIGDIR/small/743385v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@2fbb13org.highwire.dtl.DTLVardef@1a315eforg.highwire.dtl.DTLVardef@1f525bborg.highwire.dtl.DTLVardef@1801f25_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Influence of phosphate activation chemistry on the selection of the primordial genetic alphabet

RNA copying under mild conditions compatible with protocell integrity requires the input of chemical energy to drive the synthesis of activated nucleotides such as phosphorimidazolides. Recently, two potentially prebiotic classes of phosphate-activating agents have been explored, one based on isonitrile-aldehyde chemistry, the other on imine diimidazole (IDI)-N-cyanoimidazole (NCI) chemistry. Because such highly electrophilic activating agents may lead to undesirable nucleotide modifications, we have examined the reaction of both types of activating agents with the canonical ribonucleotides A, U, C, and G, and the potentially primordial nucleotides 2-thio-C (s2C), 2-thio-U (s2U), and inosine (I). We find that the isonitrile-aldehyde system shows minimal hydroxyl modification but does modify the nucleobases of U, G, s2U, and I. Except for guanosine, these modifications are readily reversible. In contrast, IDI-NCI systems acylate ribonucleotide hydroxyls while modifying nucleobases only transiently; mildly acidic pH suppresses undesired modifications. Both classes of activating agents modify 2-thiopyrimidines on the sulfur, with the isonitrile-aldehyde reaction promoting desulfurization and thus conversion to the canonical pyrimidines. To evaluate compatibility with model protocells, we tested the effects of activation chemistry on fatty acid vesicles and found that protocell integrity was preserved at moderate reagent concentrations. Our findings show that the potentially primordial s2U, s2C, and I nucleotides are more sensitive to modification than the canonical U, C, and G nucleotides, potentially contributing to the chemical selection of the early genetic alphabet.

biochemistry↗

bioRxiv2025

Nanopore-based profiling of PEGylation in nucleic acid therapeutics

Nucleic acid therapeutics, including aptamers, offer effective strategies for programmable and targeted disease treatment. To improve their stability and circulation time, oligonucleotides are often conjugated to hydrophilic polymers such as polyethylene glycol (PEG). However, current bulk techniques fail to resolve PEG heterogeneity, especially in complex biological environments. Here, we use nanopore sensing to quantify PEG conjugation efficiency of the FDA-approved RNA aptamer pegaptanib. We assembled DNA nanostructures that bind pegaptanib and then we used solid-state nanopores to quantify pegaptanib PEGylation. We further assess pegaptanib PEGylation and stability in a serum background and demonstrate that nanopore sensing resolves PEG moieties of distinct molecular weights within oligonucleotide conjugates. Single-molecule profiling of polymer-RNA conjugates enables iterative improvements in oligonucleotide design and provides a direct means to assess their stability in complex biological environments, thereby advancing the development of more effective nucleic acid therapeutics.

bioengineering↗

bioRxiv2025

A nucleic acid labeling chemistry reveals surface DNA on exosomes

Chemical labeling of nucleic acids is essential to pinpoint the structure, localization, and function of RNA and DNA. Yet, reversible sequence-independent chemistries that can label native RNA and DNA remain poorly developed. Here we describe Reversible Uridine Nitrilium-mediated Addition (RUNA), a reversible covalent chemistry that selectively modifies uridine and thymidine residues via N3 deprotonation and reaction with a nitrilium ion intermediate generated from an aldehyde and an isonitrile. The reaction forms a stable N3 adduct that can be quantitatively reversed by hydrolysis. By using reagents that are either membrane permeable or impermeable, we demonstrate the localization and function of DNA on exosomes. Although exosomes harbor nucleic acids, whether the latter are encapsulated in the exosome lumen or are surface-adhered is unknown. RUNA revealed that exosomes display DNA on their outer surface. The abundance of such surface DNA increases upon DNA-damage accumulation in cancer cells that are treated with a PARP inhibitor. This surface DNA drives exosome uptake by M2-polarized macrophages through scavenger receptors and triggers a shift toward an M1-like pro-inflammatory state. The selective labeling of surface DNA revealed an unexpected mechanism by which exosomes engage innate immune cells. RUNA is a versatile tool to analyze the nucleic acid content and functionality of extracellular vesicles in health and disease. Significance StatementPinpointing the localization of RNA and DNA in cells and organelles is central to deriving insights into their biological functions in health and disease. We describe a new method, RUNA, for labeling nucleic acids that is sequence-independent and reversible. By varying RUNA reagents, we can distinguish between nucleic acids that are located either inside or outside of membrane compartments. Using RUNA, we showed that DNA is associated with the outer surface of exosomes that are secreted by cancer cells. Further, the amount of surface DNA increases when the cancer cells are treated with an anti-cancer drug. This surface DNA promotes the uptake of exosomes by innate immune cells known as macrophages and modulates their inflammatory response.

biochemistry↗

bioRxiv2025

Impact of 2'-deoxyribo-purine substrates on nonenzymatic RNA template-directed primer extension

The composition of the primordial genetic material remains uncertain. Studies of duplex structure and stability, and of nonenzymatic template copying chemistry, provide insight into the viability of potentially primordial genetic polymers. Recent work suggests that 2'- deoxyribo-purine nucleotides may have been generated together with ribonucleotides on the early Earth. Since DNA/RNA duplexes are known to be less stable than RNA/RNA duplexes, we have examined the impact of dA, dI, and dG substitutions on RNA structure and nonenzymatic template copying. We find that single 2'-deoxyribo-purine substitutions reduce RNA duplex stability, as expected. Crystallographic studies show that such substitutions lead to minimal structural changes but point to diminished solvation as a likely reason for duplex destabilization. Kinetic studies show that dI and dG substrates exhibit slightly weaker template binding and slower rates of template-directed primer extension than the corresponding ribo-purine substrates. In contrast, dA substrates exhibit much slower reaction kinetics but higher template affinity than rA substrates. Our results suggest that a mixed RNA/DNA primordial genetic polymer would have suffered from moderately slower rates of template copying, but that this could have been offset by an advantage due to more facile strand separation or exchange. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/673048v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@15bd09aorg.highwire.dtl.DTLVardef@dc49d4org.highwire.dtl.DTLVardef@730d20org.highwire.dtl.DTLVardef@162b84f_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Potentially Prebiotic Isocyanide Activation Chemistry Drives RNA Assembly via both Nonenzymatic and Ribozyme-catalyzed Ligation

Nonenzymatic assembly of activated RNA building blocks, such as phosphorimidazolides, would have been essential for the emergence of ribozymes on the early Earth. We previously showed that ribonucleoside monophosphates can be activated to phosphorimidazolides via a prebiotically relevant phospho-Passerini reaction involving 2-aminoimidazole, 2-methylbutyraldehyde, and methyl isocyanide, and that these activated nucleotides enable template-directed nonenzymatic RNA polymerization in the same reaction mixture. Here, we demonstrate that the same chemistry activates oligoribonucleotides and drives both nonenzymatic and ribozyme-catalyzed RNA ligation within the same reaction environment. By demonstrating a continuous path from prebiotic activation chemistry to RNA template copying by both nonenzymatic and ribozyme-catalyzed ligation, our results provide a more integrated and realistic model for RNA assembly on the early Earth.

biochemistry↗

bioRxiv2025

A ribozyme ligase that requires a 3' terminal phosphate on its RNA substrate

Ribozymes played essential roles in catalyzing metabolic processes and facilitating genome replication in primordial RNA-based life. In vitro evolution has enabled the discovery of ribozymes that catalyze diverse chemical reactions, but it is likely that many catalytic activities of RNA remain to be uncovered. Expanding our understanding of the biochemical capabilities of RNA, especially new ribozyme functions related to RNA assembly, will help to refine models of primordial genome replication and, more broadly, the origin and evolution of early life. Here, we report the discovery of a novel ribozyme ligase that catalyzes the attack of the 2'-hydroxyl group of an RNA substrate on the 5'-triphosphate of a second RNA, but only when the substrate RNA possesses a 3'-phosphate vicinal to its nucleophilic 2'-hydroxyl group. This activity emerged unexpectedly during a directed evolution experiment designed to isolate ribozyme ligases that use 5'-triphosphorylated RNA oligonucleotides as substrates. Because the 3'-phosphate group on the RNA substrate does not directly participate in the ligation reaction, it would be extremely challenging to design a selection strategy to isolate ribozyme ligases with this unique reactivity. The ligases requirement for an RNA 3'-phosphate group on the substrate resembles enzymatic mechanisms found in protein-based RNA repair pathways. We propose that ribozyme-catalyzed ligation of 3'-phosphorylated RNA could have enabled the repair of cleaved RNA strands in primordial cells. We demonstrate that our in vitro evolved ribozymes ligate themselves specifically to 3'-phosphorylated RNA fragments present in heterogenous mixtures of cellular RNA, demonstrating their potential utility as enrichment reagents for profiling RNA cleavage products in transcriptomic studies. Our findings not only report a new catalytic reactivity in RNA but also provide insights into ribozyme evolution, primordial RNA repair, and potential applications in RNA sequencing.

biochemistry↗

bioRxiv2025

Nonenzymatic RNA copying with a potentially primordial genetic alphabet

Nonenzymatic RNA copying is thought to have been responsible for the replication of genetic information during the origin of life. However, chemical copying with the canonical nucleotides (A, U, G, and C) strongly favors the incorporation of G and C and disfavors the incorporation of A and especially U, because of the stronger G:C vs. A:U base pair, and the weaker stacking interactions of U. Recent advances in prebiotic chemistry suggest that the 2-thiopyrimidines were precursors to the canonical pyrimidines, raising the possibility that they may have played an important early role in RNA copying chemistry. Furthermore, 2-thiouridine (s2U) and inosine (I) form by deamination of 2-thiocytidine (s2C) and A respectively. We used thermodynamic and crystallographic analyses to compare the I:s2C and A:s2U base pairs. We find that the I:s2C base pair is isomorphic and isoenergetic with the A:s2U base pair. The I:s2C base pair is weaker than a canonical G:C base pair, while the A:s2U base pair is stronger than the canonical A:U base pair, so that a genetic alphabet consisting of s2U, s2C, I and A generates RNA duplexes with uniform base pairing energies. Consistent with these results, kinetic analysis of nonenzymatic template-directed primer extension reactions reveals that s2C and s2U substrates bind similarly to I and A in the template, and vice versa. Our work supports the plausibility of a potentially primordial genetic alphabet consisting of s2U, s2C, I and A, and offers a potential solution to the long-standing problem of biased nucleotide incorporation during nonenzymatic template copying. Significance StatementA long-standing challenge in primordial nonenzymatic RNA copying chemistry is the biased incorporation of C and G over A and U due to differences in base pair strength. We hypothesized that 2-thiopyrimidine substitution could help overcome this bias since A:s2U is a stronger version of the A:U base pair, and I:s2C is a weaker version of the G:C base pair. This study explores the efficacy of a potentially primordial genetic alphabet consisting of s2U, s2C, A and I. Our results show that A:s2U and I:s2C pairs are isoenergetic and isomorphic. Our findings highlight the potential of this alternative genetic alphabet to yield a more balanced incorporation of all nucleotides, facilitating information propagation by nonenzymatic RNA copying during the origin of life.

biochemistry↗

bioRxiv2024

Stereoselectivity of Aminoacyl-RNA Loop-closing Ligation

The origin of amino acid homochirality remains an unresolved question in the origin of life. The requirement of enantiopure nucleotides for nonenzymatic RNA copying strongly suggests that homochirality of nucleotides and RNA arose early. However, this leaves open the question of whether and how homochiral RNA subsequently imposed biological homochirality on other metabolites including amino acids. Previous studies have reported moderate stereoselectivity for various aminoacyl-RNA transfer reactions. Here we examine aminoacyl-RNA loop-closing ligation, a reaction that captures aminoacylated RNA in a stable phosphoramidate product, such that the amino acid bridges two nucleotides in the RNA backbone. We find that the rate of this reaction is much higher for RNA aminoacylated with L-amino acids than D-amino acids. We present an RNA sequence that near-exclusively captures L-amino acids in loop-closing ligation. Finally, we demonstrate that ligation of aminoacyl-L-RNA results in inverse stereoselectivity for D-amino acids. The observed stereochemical link between D-RNA and L-amino acids in the synthesis of RNA stem-loops containing bridging amino acids constitutes a stereoselective structure building process. We suggest that this process led to a selection for the evolution of aminoacyl-RNA synthetase ribozymes that were selective for L-amino acids, thereby setting the stage for the subsequent evolution of homochiral peptide and ultimately protein synthesis.

biochemistry↗

bioRxiv2024

Nanopore sequencing of intact aminoacylated tRNAs

Transfer RNAs (tRNA) are decorated during biogenesis with a variety of modifications that modulate their stability, aminoacylation, and decoding potential during translation. The complex landscape of tRNA modification presents significant analysis challenges and to date no single approach enables the simultaneous measurement of important but disparate chemical properties of individual, mature tRNA molecules. We developed a new, integrated approach to analyze the sequence, modification, and aminoacylation state of tRNA molecules in a high throughput nanopore sequencing experiment, leveraging a chemical ligation that embeds the charged amino acid in an adapted tRNA molecule. During nanopore sequencing, the embedded amino acid generates unique distortions in ionic current and translocation speed, enabling application of machine learning approaches to classify charging status and amino acid identity. Specific applications of the method indicate it will be broadly useful for examining relationships and dependencies between tRNA sequence, modification, and aminoacylation.

molecular biology↗

bioRxiv2024

Autocatalytic assembly of a chimeric aminoacyl-RNA synthetase ribozyme

Autocatalytic reactions driving the self-assembly of biological polymers are important for the origin of life, yet few experimental examples of such reactions exist. Here we report an autocatalytic assembly pathway that generates a chimeric, amino acid-bridged aminoacyl-RNA synthetase ribozyme. The noncovalent complex of ribozyme fragments initiates low level aminoacylation of one of the fragments, which after loop-closing ligation generates a highly active covalently-linked chimeric ribozyme. The generation of this ribozyme is increasingly efficient over time due to the autocatalytic assembly cycle that sustains the ribozyme over indefinite cycles of serial dilution. Due to its trans activity, this ribozyme also assembles ribozymes distinct from itself, such as the hammerhead, suggesting that RNA aminoacylation, coupled with nonenzymatic ligation, could have facilitated the emergence and propagation of ribozymes.

biochemistry↗

bioRxiv2024

Overcoming nucleotide bias in the nonenzymatic copying of RNA templates.

The RNA World hypothesis posits that RNA was the molecule of both heredity and function during the emergence of life. This hypothesis implies that RNA templates can be copied, and ultimately replicated, without the catalytic aid of evolved enzymes. A major problem with nonenzymatic templated polymerization has been the very poor copying of sequences containing rA and rU. Here we overcome that problem by using a prebiotically plausible mixture of RNA mononucleotides and random-sequence oligonucleotides, all activated by methyl isocyanide chemistry, that direct the uniform copying of arbitrary-sequence templates, including those harboring rA and rU. We further show that the use of this mixture in copying reactions suppresses copying errors while also generating a more uniform distribution of mismatches than observed for simpler systems. We find that oligonucleotide competition for template binding sites, oligonucleotide ligation, and the template binding properties of reactant intermediates work together to reduce product sequence bias and errors. Finally, we show that iterative cycling of the activation chemistry and templated polymerization improves the yield of random-sequence products. These results for random-sequence template copying are a significant advance in the pursuit of nonenzymatic RNA replication.

molecular biology↗

bioRxiv2024

Evolution of the substrate specificity of an RNA ligase ribozyme from phosphorimidazole- to triphosphate-activation

The acquisition of new RNA functions through evolutionary processes would have been essential for the diversification of RNA-based primordial biology and its subsequent transition to modern biology. However, the mechanisms by which RNAs access new functions remain unclear. Do ribozymes need completely new folds to support new but related functions, or is re-optimization of the active site sufficient? What are the roles of neutral and adaptive mutations in evolutionary innovation? Here we address these questions experimentally by focusing on the evolution of substrate specificity in RNA-catalyzed RNA assembly reactions. We use directed in vitro evolution to show that a ligase ribozyme that uses prebiotically relevant 5'-phosphorimidazole-activated substrates can be evolved to catalyze ligation with substrates that are 5'-activated with the biologically relevant triphosphate group. Interestingly, despite catalyzing a related reaction, the new ribozyme folds into a completely new structure and exhibits promiscuity by catalyzing RNA ligation with both triphosphate and phosphorimidazole-activated substrates. Although distinct in sequence and structure, the parent phosphorimidazolide ligase and the evolved triphosphate ligase ribozymes can be connected by a series of point mutations where the intermediate sequences retain at least some ligase activity. The existence of a quasi-neutral pathway between these distinct ligase ribozymes suggests that neutral drift is sufficient to enable the acquisition of new substrate specificity, thereby providing opportunities for subsequent adaptive optimization. The transition from RNA-catalyzed RNA assembly using phosphorimidazole-activated substrates to triphosphate-activated substrates may have set the stage for the later evolution of the protein enzymes that use monomeric triphosphates (NTPs) for RNA synthesis.

biochemistry↗

bioRxiv2024

Structure-guided aminoacylation and assembly of chimeric RNAs

Coded ribosomal peptide synthesis could not have evolved unless its sequence and amino acid specific aminoacylated tRNA substrates already existed. We therefore wondered whether aminoacylated RNAs might have served some primordial function prior to their role in protein synthesis. Here we show that specific RNA sequences can be nonenzymatically aminoacylated and ligated to produce amino acid-bridged stem-loop RNAs. We used deep sequencing to identify RNAs that undergo highly efficient glycine aminoacylation followed by loop-closing ligation. The crystal structure of one such glycine-bridged RNA hairpin reveals a compact internally stabilized structure with the same eponymous T-loop architecture found in modern tRNA. We demonstrate that the T-loop assisted amino acid bridging of RNA oligonucleotides enables the rapid template-free assembly of a chimeric version of an aminoacyl-RNA synthetase ribozyme. We suggest that the primordial assembly of such chimeric ribozymes would have allowed the greater functionality of amino acids to contribute to enhanced ribozyme catalysis, providing a driving force for the evolution of sequence and amino acid specific aminoacyl-RNA synthetase enzymes prior to their role in protein synthesis.

biochemistry↗

bioRxiv2024

Diaminopurine in Nonenzymatic RNA Template Copying

In the primordial RNA World, before the advent of ribozymes, nonenzymatic template copying would have been essential for the transmission of genetic information. However, the products of chemical copying with the canonical nucleotides are heavily biased towards the incorporation of G and C. Diaminopurine (D) can form a D:U base pair that is almost as strong as a G:C base pair. We therefore asked whether replacing A with D might lead to more efficient and less biased nonenzymatic template copying. As expected, primer extension substrates containing D bind 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. To investigate the nature of the D:C mismatch, we solved the crystal structure of RNA duplexes containing D:C mismatches, and showed that D can form a wobble-type base pair with C. We then asked whether competition with G would decrease 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 diaminopurine deserves further attention for its possible role in the RNA World, and as a potentially useful component of artificial nonenzymatic RNA replication systems.

biochemistry↗

bioRxiv2024

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 a series of 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 with mixtures of nucleotides confirmed the bias against the incorporation of threo-nucleotides into oligonucleotides. This bias, coupled with selective prebiotic synthesis and templated copying favoring ribonucleotides, provides a plausible model for the exclusion of threo-nucleotides from primordial oligonucleotides. In contrast, the exclusion of arabino-nucleotides may have resulted primarily from biases in synthesis and in template-directed primer extension.

biochemistry↗

bioRxiv2023

Transient States During Oligonucleotides Hybridization: Insights from Annealing Kinetics of Mismatches and Bulges

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 12bp 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 vs. 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.

biochemistry↗

bioRxiv2023

Molecular crowding facilitates ribozyme-catalyzed RNA assembly

Catalytic RNAs or ribozymes are considered to be central to primordial biology. Most ribozymes require moderate to high concentrations of divalent cations such as Mg2+ to fold into their catalytically competent structures and perform catalysis. However, undesirable effects of Mg2+ such as hydrolysis of reactive RNA building blocks and degradation of RNA structures are likely to undermine its beneficial roles in ribozyme catalysis. Further, prebiotic cell-like compartments bounded by fatty acid membranes are destabilized in the presence of Mg2+, making ribozyme function inside prebiotically relevant protocells a significant challenge. Therefore, we sought to identify conditions that would enable ribozymes to retain activity in low concentrations of Mg2+. Inspired by the ability of ribozymes to function inside crowded cellular environments with <1 mM free Mg2+, we tested molecular crowding as a potential mechanism to lower the Mg2+ concentration required for ribozyme-catalyzed RNA assembly. Here, we show that the ribozyme-catalyzed ligation of phosphorimidazolide RNA substrates is significantly enhanced in the presence of the artificial crowding agent polyethylene glycol. We also found that molecular crowding preserves ligase activity under denaturing conditions such as alkaline pH and the presence of urea. We also show that crowding-induced stimulation of RNA-catalyzed RNA assembly is not limited to phosphorimidazolide ligation but extends to the RNA-catalyzed polymerization of nucleoside triphosphates. RNA-catalyzed RNA ligation is also stimulated by the presence of prebiotically relevant small molecules such as ethylene glycol, ribose, and amino acids, consistent with a role for molecular crowding in primordial ribozyme function and more generally, in the emergence of RNA-based cellular life. Table of Contents Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=94 SRC="FIGDIR/small/538884v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@17d282borg.highwire.dtl.DTLVardef@1d8531aorg.highwire.dtl.DTLVardef@1380f7borg.highwire.dtl.DTLVardef@180708f_HPS_FORMAT_FIGEXP M_FIG C_FIG SYNOPSISMolecular crowding enables ribozyme-catalyzed RNA assembly under low Mg2+ conditions, and consequently may have played an essential role in the origin and evolution of RNA-based primordial life.

biochemistry↗