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Jack W Szostak - One of the best experts on this subject based on the ideXlab platform.
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competition between bridged dinucleotides and activated mononucleotides determines the error frequency of nonenzymatic rna Primer Extension
Nucleic Acids Research, 2021Co-Authors: Daniel Duzdevich, Christopher E Carr, Travis Walton, Dian Ding, Stephanie J Zhang, Jack W SzostakAbstract: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.
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deep sequencing of non enzymatic rna Primer Extension
Nucleic Acids Research, 2020Co-Authors: Daniel Duzdevich, Christopher E Carr, Jack W SzostakAbstract:Life emerging in an RNA world is expected to propagate RNA as hereditary information, requiring some form of primitive replication without enzymes. Non-enzymatic template-directed RNA Primer Extension is a model of the copying step in this posited form of replication. The sequence space accessed by Primer Extension dictates potential pathways to self-replication and, eventually, ribozymes. Which sequences can be accessed? What is the fidelity of the reaction? Does the recently illuminated mechanism of Primer Extension affect the distribution of sequences that can be copied? How do sequence features respond to experimental conditions and prebiotically relevant contexts? To help answer these and related questions, we here introduce a deep-sequencing methodology for studying RNA Primer Extension. We have designed and vetted special RNA constructs for this purpose, honed a protocol for sample preparation and developed custom software that analyzes sequencing data. We apply this new methodology to proof-of-concept controls, and demonstrate that it works as expected and reports on key features of the sequences accessed by Primer Extension.
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deep sequencing of nonenzymatic rna Primer Extension
bioRxiv, 2020Co-Authors: Christopher E Carr, Daniel Duzdevich, Jack W SzostakAbstract: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.
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non enzymatic Primer Extension with strand displacement
eLife, 2019Co-Authors: Constantin Giurgiu, Derek K Oflaherty, Lijun Zhou, Seohyun Chris Kim, Tom H Wright, Jack W SzostakAbstract:Non-enzymatic RNA self-replication is integral to the emergence of the 'RNA World'. Despite considerable progress in non-enzymatic template copying, demonstrating a full replication cycle remains challenging due to the difficulty of separating the strands of the product duplex. Here, we report a prebiotically plausible approach to strand displacement synthesis in which short 'invader' oligonucleotides unwind an RNA duplex through a toehold/branch migration mechanism, allowing non-enzymatic Primer Extension on a template that was previously occupied by its complementary strand. Kinetic studies of single-step reactions suggest that following invader binding, branch migration results in a 2:3 partition of the template between open and closed states. Finally, we demonstrate continued Primer Extension with strand displacement by employing activated 3'-aminonucleotides, a more reactive proxy for ribonucleotides. Our study suggests that complete cycles of non-enzymatic replication of the primordial genetic material may have been facilitated by short RNA oligonucleotides.
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non enzymatic Primer Extension with strand displacement
bioRxiv, 2019Co-Authors: Constantin Giurgiu, Derek K Oflaherty, Lijun Zhou, Seohyun Chris Kim, Tom H Wright, Jack W SzostakAbstract:Abstract Non-enzymatic RNA self-replication is integral to the ‘RNA World’ hypothesis. Despite considerable progress in non-enzymatic template copying, true replication remains challenging due to the difficulty of separating the strands of the product duplex. Here, we report a prebiotically plausible solution to this problem in which short ‘invader’ oligonucleotides unwind an RNA duplex through a toehold/branch migration mechanism, allowing non-enzymatic Primer Extension on a template that was previously occupied by its complementary strand. Kinetic studies of single-step reactions suggest that following invader binding, branch migration results in a 2:3 partition of the template between open and closed states. Finally, we demonstrate continued Primer Extension with strand displacement by employing activated 3′-aminonucleotides, a more reactive proxy for ribonucleotides. Our study suggests that complete cycles of non-enzymatic replication of the primordial genetic material may have been catalyzed by short RNA oligonucleotides.
Kevin M Weeks - One of the best experts on this subject based on the ideXlab platform.
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selective 2 hydroxyl acylation analyzed by Primer Extension and mutational profiling shape map for direct versatile and accurate rna structure analysis
Nature Protocols, 2015Co-Authors: Matthew J Smola, Greggory M Rice, Steven Busan, Nathan A Siegfried, Kevin M WeeksAbstract:Selective 2'-hydroxyl acylation analyzed by Primer Extension (SHAPE) chemistries exploit small electrophilic reagents that react with 2'-hydroxyl groups to interrogate RNA structure at single-nucleotide resolution. Mutational profiling (MaP) identifies modified residues by using reverse transcriptase to misread a SHAPE-modified nucleotide and then counting the resulting mutations by massively parallel sequencing. The SHAPE-MaP approach measures the structure of large and transcriptome-wide systems as accurately as can be done for simple model RNAs. This protocol describes the experimental steps, implemented over 3 d, that are required to perform SHAPE probing and to construct multiplexed SHAPE-MaP libraries suitable for deep sequencing. Automated processing of MaP sequencing data is accomplished using two software packages. ShapeMapper converts raw sequencing files into mutational profiles, creates SHAPE reactivity plots and provides useful troubleshooting information. SuperFold uses these data to model RNA secondary structures, identify regions with well-defined structures and visualize probable and alternative helices, often in under 1 d. SHAPE-MaP can be used to make nucleotide-resolution biophysical measurements of individual RNA motifs, rare components of complex RNA ensembles and entire transcriptomes.
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selective 2 hydroxyl acylation analyzed by Primer Extension shape quantitative rna structure analysis at single nucleotide resolution
Nature Protocols, 2006Co-Authors: Kevin A Wilkinson, Edward J Merino, Kevin M WeeksAbstract:Selective 2'-hydroxyl acylation analyzed by Primer Extension (SHAPE) interrogates local backbone flexibility in RNA at single-nucleotide resolution under diverse solution environments. Flexible RNA nucleotides preferentially sample local conformations that enhance the nucleophilic reactivity of 2'-hydroxyl groups toward electrophiles, such as N-methylisatoic anhydride (NMIA). Modified sites are detected as stops in an optimized Primer Extension reaction, followed by electrophoretic fragment separation. SHAPE chemistry scores local nucleotide flexibility at all four ribonucleotides in a single experiment and discriminates between base-paired versus unconstrained or flexible residues with a dynamic range of 20-fold or greater. Quantitative SHAPE reactivity information can be used to establish the secondary structure of an RNA, to improve the accuracy of structure prediction algorithms, to monitor structural differences between related RNAs or a single RNA in different states, and to detect ligand binding sites. SHAPE chemistry rarely needs significant optimization and requires two days to complete for an RNA of 100-200 nucleotides.
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selective 2 hydroxyl acylation analyzed by Primer Extension shape quantitative rna structure analysis at single nucleotide resolution
Nature Protocols, 2006Co-Authors: Kevin A Wilkinson, Edward J Merino, Kevin M WeeksAbstract:Selective 2′-hydroxyl acylation analyzed by Primer Extension (SHAPE) interrogates local backbone flexibility in RNA at single-nucleotide resolution under diverse solution environments. Flexible RNA nucleotides preferentially sample local conformations that enhance the nucleophilic reactivity of 2′-hydroxyl groups toward electrophiles, such as N-methylisatoic anhydride (NMIA). Modified sites are detected as stops in an optimized Primer Extension reaction, followed by electrophoretic fragment separation. SHAPE chemistry scores local nucleotide flexibility at all four ribonucleotides in a single experiment and discriminates between base-paired versus unconstrained or flexible residues with a dynamic range of 20-fold or greater. Quantitative SHAPE reactivity information can be used to establish the secondary structure of an RNA, to improve the accuracy of structure prediction algorithms, to monitor structural differences between related RNAs or a single RNA in different states, and to detect ligand binding sites. SHAPE chemistry rarely needs significant optimization and requires two days to complete for an RNA of 100–200 nucleotides. Note: In the version of this article initially published online, the value for the pH range in the second paragraph on page 2 was incorrect; it should have read 7.5–8.2. In addition, two sentences were included that should have been removed from page 1. These errors have been corrected in all versions of the article.
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rna structure analysis at single nucleotide resolution by selective 2 hydroxyl acylation and Primer Extension shape
Journal of the American Chemical Society, 2005Co-Authors: Edward J Merino, Kevin A Wilkinson, Jennifer L Coughlan, Kevin M WeeksAbstract:The reactivity of an RNA ribose hydroxyl is shown to be exquisitely sensitive to local nucleotide flexibility because a conformationally constrained adjacent 3‘-phosphodiester inhibits formation of the deprotonated, nucleophilic oxyanion form of the 2‘-hydroxyl group. Reaction with an appropriate electrophile, N-methylisatoic anhydride, to form a 2‘-O-adduct thus can be used to monitor local structure at every nucleotide in an RNA. We develop a quantitative approach involving Selective 2‘-Hydroxyl Acylation analyzed by Primer Extension (SHAPE) to map the structure of and to distinguish fine differences in structure for tRNAAsp transcripts at single nucleotide resolution. Modest Extensions of the SHAPE approach will allow RNA structure to be monitored comprehensively and at single nucleotide resolution for RNAs of arbitrary sequence and structural complexity and under diverse solution environments.
Kevin A Wilkinson - One of the best experts on this subject based on the ideXlab platform.
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selective 2 hydroxyl acylation analyzed by Primer Extension shape quantitative rna structure analysis at single nucleotide resolution
Nature Protocols, 2006Co-Authors: Kevin A Wilkinson, Edward J Merino, Kevin M WeeksAbstract:Selective 2′-hydroxyl acylation analyzed by Primer Extension (SHAPE) interrogates local backbone flexibility in RNA at single-nucleotide resolution under diverse solution environments. Flexible RNA nucleotides preferentially sample local conformations that enhance the nucleophilic reactivity of 2′-hydroxyl groups toward electrophiles, such as N-methylisatoic anhydride (NMIA). Modified sites are detected as stops in an optimized Primer Extension reaction, followed by electrophoretic fragment separation. SHAPE chemistry scores local nucleotide flexibility at all four ribonucleotides in a single experiment and discriminates between base-paired versus unconstrained or flexible residues with a dynamic range of 20-fold or greater. Quantitative SHAPE reactivity information can be used to establish the secondary structure of an RNA, to improve the accuracy of structure prediction algorithms, to monitor structural differences between related RNAs or a single RNA in different states, and to detect ligand binding sites. SHAPE chemistry rarely needs significant optimization and requires two days to complete for an RNA of 100–200 nucleotides. Note: In the version of this article initially published online, the value for the pH range in the second paragraph on page 2 was incorrect; it should have read 7.5–8.2. In addition, two sentences were included that should have been removed from page 1. These errors have been corrected in all versions of the article.
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selective 2 hydroxyl acylation analyzed by Primer Extension shape quantitative rna structure analysis at single nucleotide resolution
Nature Protocols, 2006Co-Authors: Kevin A Wilkinson, Edward J Merino, Kevin M WeeksAbstract:Selective 2'-hydroxyl acylation analyzed by Primer Extension (SHAPE) interrogates local backbone flexibility in RNA at single-nucleotide resolution under diverse solution environments. Flexible RNA nucleotides preferentially sample local conformations that enhance the nucleophilic reactivity of 2'-hydroxyl groups toward electrophiles, such as N-methylisatoic anhydride (NMIA). Modified sites are detected as stops in an optimized Primer Extension reaction, followed by electrophoretic fragment separation. SHAPE chemistry scores local nucleotide flexibility at all four ribonucleotides in a single experiment and discriminates between base-paired versus unconstrained or flexible residues with a dynamic range of 20-fold or greater. Quantitative SHAPE reactivity information can be used to establish the secondary structure of an RNA, to improve the accuracy of structure prediction algorithms, to monitor structural differences between related RNAs or a single RNA in different states, and to detect ligand binding sites. SHAPE chemistry rarely needs significant optimization and requires two days to complete for an RNA of 100-200 nucleotides.
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rna structure analysis at single nucleotide resolution by selective 2 hydroxyl acylation and Primer Extension shape
Journal of the American Chemical Society, 2005Co-Authors: Edward J Merino, Kevin A Wilkinson, Jennifer L Coughlan, Kevin M WeeksAbstract:The reactivity of an RNA ribose hydroxyl is shown to be exquisitely sensitive to local nucleotide flexibility because a conformationally constrained adjacent 3‘-phosphodiester inhibits formation of the deprotonated, nucleophilic oxyanion form of the 2‘-hydroxyl group. Reaction with an appropriate electrophile, N-methylisatoic anhydride, to form a 2‘-O-adduct thus can be used to monitor local structure at every nucleotide in an RNA. We develop a quantitative approach involving Selective 2‘-Hydroxyl Acylation analyzed by Primer Extension (SHAPE) to map the structure of and to distinguish fine differences in structure for tRNAAsp transcripts at single nucleotide resolution. Modest Extensions of the SHAPE approach will allow RNA structure to be monitored comprehensively and at single nucleotide resolution for RNAs of arbitrary sequence and structural complexity and under diverse solution environments.
Travis Walton - One of the best experts on this subject based on the ideXlab platform.
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competition between bridged dinucleotides and activated mononucleotides determines the error frequency of nonenzymatic rna Primer Extension
Nucleic Acids Research, 2021Co-Authors: Daniel Duzdevich, Christopher E Carr, Travis Walton, Dian Ding, Stephanie J Zhang, Jack W SzostakAbstract: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.
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template directed catalysis of a multistep reaction pathway for nonenzymatic rna Primer Extension
Biochemistry, 2019Co-Authors: Travis Walton, Jack W Szostak, Lydia PazienzaAbstract:Before the advent of polymerase enzymes, the copying of genetic material during the origin of life may have involved the nonenzymatic polymerization of RNA monomers that are more reactive than the biological nucleoside triphosphates. Activated RNA monomers such as nucleotide 5'-phosphoro-2-aminoimidazolides spontaneously form an imidazolium-bridged dinucleotide intermediate that undergoes rapid nonenzymatic template-directed Primer Extension. However, it is unknown whether the intermediate can form on the template or only in solution and whether the intermediate is prone to hydrolysis when bound to the template or reacts preferentially with the Primer. Here we show that an activated monomer can first bind the template and then form an imidazolium-bridged intermediate by reacting with a 2-aminoimidazole-activated downstream oligonucleotide. We have also characterized the partition of the template-bound intermediate between hydrolysis and Primer Extension. In the presence of the catalytic metal ion Mg2+, >90% of the template-bound intermediate reacts with the adjacent Primer to generate the Primer Extension product while less than 10% reacts with competing water. Our results indicate that an RNA template can catalyze a multistep phosphodiester bond formation pathway while minimizing hydrolysis with a specificity reminiscent of an enzyme-catalyzed reaction.
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synthesis of a nonhydrolyzable nucleotide phosphoroimidazolide analogue that catalyzes nonenzymatic rna Primer Extension
Journal of the American Chemical Society, 2018Co-Authors: Chun Pong Tam, Lijun Zhou, Wen Zhang, Albert C Fahrenbach, Travis WaltonAbstract:We report the synthesis of guanosine 5'-(4-methylimidazolyl)phosphonate (ICG), the third member of a series of nonhydrolyzable nucleoside 5'-phosphoro-2-methylimidazolide (2-MeImpN) analogues designed for mechanistic studies of nonenzymatic RNA Primer Extension. The addition of a 2-MeImpN monomer to a Primer is catalyzed by the presence of a downstream activated monomer, yet the three nonhydrolyzable analogues do not show catalytic effects under standard mildly basic Primer Extension conditions. Surprisingly, ICG, which has a pKa similar to that of 2-MeImpG, is a modest catalyst of nonenzymatic Primer Extension at acidic pH. Here we show that ICG reacts with 2-MeImpC to form a stable 5'-5'-imidazole-bridged guanosine-cytosine dinucleotide, with both a labile nitrogen-phosphorus and a stable carbon-phosphorus linkage flanking the central imidazole bridge. Cognate RNA Primer-template complexes react with this GC-dinucleotide by attack of the Primer 3'-hydroxyl on the activated N-P side of the 5'-5'-imidazole bridge. These observations support the hypothesis that 5'-5'-imidazole-bridged dinucleotides can bind to cognate RNA Primer-template duplexes and adopt appropriate conformations for subsequent phosphodiester bond formation, consistent with our recent mechanistic proposal that the formation of activated 5'-5'-imidazolium-bridged dinucleotides is responsible for 2-MeImpN-driven Primer Extension.
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Template-Directed Catalysis of a Multistep Reaction Pathway for Nonenzymatic RNA Primer Extension
2018Co-Authors: Travis Walton, Lydia Pazienza, Jack W SzostakAbstract:Before the advent of polymerase enzymes, the copying of genetic material during the origin of life may have involved the nonenzymatic polymerization of RNA monomers that are more reactive than the biological nucleoside triphosphates. Activated RNA monomers such as nucleotide 5′-phosphoro-2-aminoimidazolides spontaneously form an imidazolium-bridged dinucleotide intermediate that undergoes rapid nonenzymatic template-directed Primer Extension. However, it is unknown whether the intermediate can form on the template or only in solution and whether the intermediate is prone to hydrolysis when bound to the template or reacts preferentially with the Primer. Here we show that an activated monomer can first bind the template and then form an imidazolium-bridged intermediate by reacting with a 2-aminoimidazole-activated downstream oligonucleotide. We have also characterized the partition of the template-bound intermediate between hydrolysis and Primer Extension. In the presence of the catalytic metal ion Mg2+, >90% of the template-bound intermediate reacts with the adjacent Primer to generate the Primer Extension product while less than 10% reacts with competing water. Our results indicate that an RNA template can catalyze a multistep phosphodiester bond formation pathway while minimizing hydrolysis with a specificity reminiscent of an enzyme-catalyzed reaction
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insight into the mechanism of nonenzymatic rna Primer Extension from the structure of an rna gpppg complex
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Chun Pong Tam, Travis Walton, Wen Zhang, Albert C Fahrenbach, Gabriel Birrane, Jack W SzostakAbstract:The nonenzymatic copying of RNA templates with imidazole-activated nucleotides is a well-studied model for the emergence of RNA self-replication during the origin of life. We have recently discovered that this reaction can proceed through the formation of an imidazolium-bridged dinucleotide intermediate that reacts rapidly with the Primer. To gain insight into the relationship between the structure of this intermediate and its reactivity, we cocrystallized an RNA Primer–template complex with a close analog of the intermediate, the triphosphate-bridged guanosine dinucleotide GpppG, and solved a high-resolution X-ray structure of the complex. The structure shows that GpppG binds the RNA template through two Watson–Crick base pairs, with the Primer 3ʹ-hydroxyl oriented to attack the 5ʹ-phosphate of the adjacent G residue. Thus, the GpppG structure suggests that the bound imidazolium-bridged dinucleotide intermediate would be preorganized to react with the Primer by in-line S N 2 substitution. The structures of bound GppG and GppppG suggest that the length and flexibility of the 5ʹ-5ʹ linkage are important for optimal preorganization of the complex, whereas the position of the 5ʹ-phosphate of bound pGpG explains the slow rate of oligonucleotide ligation reactions. Our studies provide a structural interpretation for the observed reactivity of the imidazolium-bridged dinucleotide intermediate in nonenzymatic RNA Primer Extension.
Chun Pong Tam - One of the best experts on this subject based on the ideXlab platform.
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structural rationale for the enhanced catalysis of nonenzymatic rna Primer Extension by a downstream oligonucleotide
Journal of the American Chemical Society, 2018Co-Authors: Wen Zhang, Chun Pong Tam, Jack W Szostak, Lijun Zhou, Jiawei WangAbstract:Nonenzymatic RNA Primer Extension by activated mononucleotides has long served as a model for the study of prebiotic RNA copying. We have recently shown that the rate of Primer Extension is greatly enhanced by the formation of an imidazolium-bridged dinucleotide between the incoming monomer and a second, downstream activated monomer. However, the rate of Primer Extension is further enhanced if the downstream monomer is replaced by an activated oligonucleotide. Even an unactivated downstream oligonucleotide provides a modest enhancement in the rate of reaction of a Primer with a single activated monomer. Here we study the mechanism of these effects through crystallographic studies of RNA complexes with the recently synthesized nonhydrolyzable substrate analog, guanosine 5′-(4-methylimidazolyl)-phosphonate (ICG). ICG mimics 2-methylimidazole activated guanosine-5′-phosphate (2-MeImpG), a commonly used substrate in nonenzymatic Primer Extension experiments. We present crystal structures of Primer-template co...
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synthesis of a nonhydrolyzable nucleotide phosphoroimidazolide analogue that catalyzes nonenzymatic rna Primer Extension
Journal of the American Chemical Society, 2018Co-Authors: Chun Pong Tam, Lijun Zhou, Wen Zhang, Albert C Fahrenbach, Travis WaltonAbstract:We report the synthesis of guanosine 5'-(4-methylimidazolyl)phosphonate (ICG), the third member of a series of nonhydrolyzable nucleoside 5'-phosphoro-2-methylimidazolide (2-MeImpN) analogues designed for mechanistic studies of nonenzymatic RNA Primer Extension. The addition of a 2-MeImpN monomer to a Primer is catalyzed by the presence of a downstream activated monomer, yet the three nonhydrolyzable analogues do not show catalytic effects under standard mildly basic Primer Extension conditions. Surprisingly, ICG, which has a pKa similar to that of 2-MeImpG, is a modest catalyst of nonenzymatic Primer Extension at acidic pH. Here we show that ICG reacts with 2-MeImpC to form a stable 5'-5'-imidazole-bridged guanosine-cytosine dinucleotide, with both a labile nitrogen-phosphorus and a stable carbon-phosphorus linkage flanking the central imidazole bridge. Cognate RNA Primer-template complexes react with this GC-dinucleotide by attack of the Primer 3'-hydroxyl on the activated N-P side of the 5'-5'-imidazole bridge. These observations support the hypothesis that 5'-5'-imidazole-bridged dinucleotides can bind to cognate RNA Primer-template duplexes and adopt appropriate conformations for subsequent phosphodiester bond formation, consistent with our recent mechanistic proposal that the formation of activated 5'-5'-imidazolium-bridged dinucleotides is responsible for 2-MeImpN-driven Primer Extension.
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a mechanistic explanation for the regioselectivity of nonenzymatic rna Primer Extension
Journal of the American Chemical Society, 2017Co-Authors: Constantin Giurgiu, Derek K Oflaherty, Chun Pong Tam, Jack W SzostakAbstract:A working model of nonenzymatic RNA Primer Extension could illuminate how prebiotic chemistry transitioned to biology. All currently known experimental reconstructions of nonenzymatic RNA Primer Extension yield a mixture of 2′-5′ and 3′-5′ internucleotide linkages. Although long seen as a major problem, the causes of the poor regioselectivity of the reaction are unknown. We used a combination of different leaving groups, nucleobases, and templating sequences to uncover the factors that yield selective formation of 3′-5′ internucleotide linkages. We found that fast and high yielding reactions selectively form 3′-5′ linkages. Additionally, in all cases with high 3′-5′ regioselectivity, Watson–Crick base pairing between the RNA monomers and the template is observed at the Extension site and at the adjacent downstream position. Mismatched base-pairs and other factors that would perturb the geometry of the imidazolium bridged intermediate lower both the rate and regioselectivity of the reaction.
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insight into the mechanism of nonenzymatic rna Primer Extension from the structure of an rna gpppg complex
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Chun Pong Tam, Travis Walton, Wen Zhang, Albert C Fahrenbach, Gabriel Birrane, Jack W SzostakAbstract:The nonenzymatic copying of RNA templates with imidazole-activated nucleotides is a well-studied model for the emergence of RNA self-replication during the origin of life. We have recently discovered that this reaction can proceed through the formation of an imidazolium-bridged dinucleotide intermediate that reacts rapidly with the Primer. To gain insight into the relationship between the structure of this intermediate and its reactivity, we cocrystallized an RNA Primer–template complex with a close analog of the intermediate, the triphosphate-bridged guanosine dinucleotide GpppG, and solved a high-resolution X-ray structure of the complex. The structure shows that GpppG binds the RNA template through two Watson–Crick base pairs, with the Primer 3ʹ-hydroxyl oriented to attack the 5ʹ-phosphate of the adjacent G residue. Thus, the GpppG structure suggests that the bound imidazolium-bridged dinucleotide intermediate would be preorganized to react with the Primer by in-line S N 2 substitution. The structures of bound GppG and GppppG suggest that the length and flexibility of the 5ʹ-5ʹ linkage are important for optimal preorganization of the complex, whereas the position of the 5ʹ-phosphate of bound pGpG explains the slow rate of oligonucleotide ligation reactions. Our studies provide a structural interpretation for the observed reactivity of the imidazolium-bridged dinucleotide intermediate in nonenzymatic RNA Primer Extension.
