A Highly Reactive Imidazolium-Bridged Dinucleotide Intermediate in Nonenzymatic RNA Primer Extension
Open accessAbstract
Because of its importance for the origin of life, the nonenzymatic copying of RNA templates has been the subject of intense study for several decades. Previous characterizations of template-directed primer extension using 5′-phosphoryl-2-methylimidazole-activated nucleotides (2-MeImpNs) as substrates have assumed a classical in-line nucleophilic substitution mechanism, in which the 3′-hydroxyl of the primer attacks the phosphate of the incoming monomer, displacing the 2-methylimidazole leaving group. However, we have found that the initial rate of primer extension depends on the pH and concentration at which the activated monomer is maintained prior to the primer extension reaction. These and other results suggest an alternative mechanism, in which two monomers react with each other to form an imidazolium-bridged dinucleotide intermediate, which then binds to the template. Subsequent attack of the 3′-hydroxyl of the primer displaces an activated nucleotide as the leaving group and results in extension of the primer by one nucleotide. Analysis of monomer solutions by NMR indicates formation of the proposed imidazolium-bridged dinucleotide in the expected pH-dependent manner. We have used synthetic methods to prepare material that is enriched in this proposed intermediate and show that it is a highly reactive substrate for primer extension. The formation of an imidazolium-bridged dinucleotide intermediate provides a mechanistic interpretation of previously observed catalysis by an activated nucleotide located downstream from the site of primer extension.
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Szostak and co-workers found that 5′-5′imidazolium bridged dinucleotides improved fidelity and reaction rates by virtue of higher template-affinity and geometrical positioning of the reactive species.
“Szostak and co-workers found that 5′-5′imidazolium bridged dinucleotides, which are formed spontaneously in the nucleotide activation reaction, improved fidelity and reaction rates by virtue of higher template-affinity and geometrical positioning of the reactive species.12−14”
From Sequencing of <scp>d</scp>/<scp>l</scp>-DNA and XNA by Templated-Synthesis (2025) · cites this paper as [12]
The Results and Discussion report that the imidazolium-bridged dinucleotide forms from activated monomers and is a highly reactive substrate that increases primer-extension rates. The Discussion proposes that its geometry may favor reaction, but says its template affinity remains to be measured; it does not establish improved copying fidelity. The claim presents those proposed mechanisms and the fidelity benefit as findings. (LLM, conf=0.88)
Experimental — accuracy not yet measuredThe intermediate could bind to the template through two adjacent Watson-Crick base pairs.
“The template specificity of primer extension suggested that the intermediate could bind to the template through two adjacent Watson-Crick base pairs”
From Protocells and RNA Self-Replication (2018) · cites this paper as Walton and Szostak 2016
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- Authors
- Walton, T., & Szostak, J. W.
- Affiliations
- Howard Hughes Medical Institute (US)
- Corresponding author
- Szostak, Jack W.
- Venue
- Journal of the American Chemical Society
- Publisher
- American Chemical Society (ACS)
- Published
- 2016-08-23
- Volume
- 138
- Issue
- 36
- Pages
- 11996-12002
- ISSN
- 0002-7863 (ISSN-L)
- 1520-5126
- PMC
- PMC6326528
- OpenAlex
- W2512565170
- Cited by
- 102
- References
- 4
- Citations by year
- 2026: 7 · 2025: 11 · 2024: 12 · 2023: 7 · 2022: 5 · 2021: 7 · 2020: 16 · 2019: 10 · 2018: 14 · 2017: 12 · 2016: 1
- FWCI
- 6.06
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Locations
- Journal of the American Chemical Society (publishedVersion, other-oa)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Simons Foundation (290363)
- Topics
- Physical Sciences › Physics and Astronomy › Astronomy and Astrophysics › Origins and Evolution of Life
- Physical Sciences › Chemistry › Organic Chemistry › Chemical Reaction Mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
- Keywords
- origin of life, nucleophilic substitution mechanism, pH dependence
- MeSH terms
- Imidazoles, Isotope Labeling, Kinetics, Models, Molecular, Nucleic Acid Conformation, Nucleotides, RNA
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