Kinetics of RNA Degradation by Specific Base Catalysis of Transesterification Involving the 2‘-Hydroxyl Group
Abstract
A detailed understanding of the susceptibility of RNA phosphodiesters to specific base-catalyzed cleavage is necessary to approximate the stability of RNA under various conditions. In addition, quantifying the rate enhancements that can be produced exclusively by this common cleavage mechanism is needed to fully interpret the mechanisms employed by ribonucleases and by RNA-cleaving ribozymes. Chimeric DNA/RNA oligonucleotides were used to examine the rates of hydroxide-dependent degradation of RNA phosphodiesters under reaction conditions that simulate those of biological systems. Under neutral or alkaline pH conditions, the dominant pathway for RNA degradation is an internal phosphoester transfer reaction that is promoted by specific base catalysis. As expected, increasing the concentration of hydroxide ion, increasing the concentration of divalent magnesium, or raising the temperature accelerates strand scission. In most instances, the identities of the nucleotide bases that flank the target RNA linkage have a negligible effect on the p K a of the nucleophilic 2‘-hydroxyl group, and only have a minor effect on the maximum rate constant for the transesterification reaction. Under representative physiological conditions, specific base catalysis of RNA cleavage generates a maximum rate enhancement of ∼100 000-fold over the background rate of RNA transesterification. The kinetic parameters reported herein provide theoretical limits for the stability of RNA polymers and for the proficiency of RNA-cleaving enzymes and enzyme mimics that exclusively employ a mechanism of general base catalysis.
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The C2'-OH group renders RNA unstable; under alkaline conditions, deprotonation generates a nucleophile that attacks the 5' phosphorous, forming a 2',3'-cyclic phosphate and 5'-OH.
“the C2′ –OH group renders RNA unstable; under alkaline conditions, deprotonation of the C2′ –OH generates a good nucleophile, which can attack the electrophilic phosphorous atom 5′ to the ribonucleotide, forming a 2′,3′-cyclic phosphate, as well as a 5′ –OH”
From Ribonucleotide errors, RNase HII, and ribonucleotide excision repair (2026) · cites this paper as Li and Breaker 1999
The nicking of longer circRNAs is likely mediated by magnesium-catalyzed autohydrolysis.
“The nicking of longer circRNAs that we observed during the production of circRNA (Supplementary Fig. 2a), likely mediated by magnesium-catalyzed autohydrolysis27, significantly reduces yields and is another deficiency that requires improvement.”
From Engineering circular RNA for potent and stable translation in eukaryotic cells (2018) · cites this paper as [27]
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“OH (Oivanen et al. 1998; Li and Breaker 1999; Caldecott 2014).”
- Authors
- Li, Y., & Breaker, R. R.
- Affiliations
- Yale University (US)
- Corresponding author
- Breaker, Ronald R.
- Venue
- Journal of the American Chemical Society
- Publisher
- American Chemical Society (ACS)
- Published
- 1999-05-25
- Volume
- 121
- Issue
- 23
- Pages
- 5364-5372
- ISSN
- 0002-7863 (ISSN-L)
- 1520-5126
- OpenAlex
- W1964261732
- Cited by
- 587
- References
- 43
- Citations by year
- 2026: 32 · 2025: 49 · 2024: 37 · 2023: 28 · 2022: 32 · 2021: 38 · 2020: 27 · 2019: 28 · 2018: 22 · 2017: 26 · 2016: 21 · 2015: 27 · 2014: 31 · 2013: 17 · 2012: 16
- FWCI
- 8.60
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Journal of the American Chemical Society (publishedVersion)
- Funders
- Division of Chemistry
- Topics
- Physical Sciences › Chemistry › Organic Chemistry › Chemical Reaction Mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA and Nucleic Acid Chemistry
- Keywords
- RNA degradation, base catalysis, transesterification, 2'-hydroxyl group, RNA stability, rate enhancement
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