Thermodynamics of RNA−RNA Duplexes with 2- or 4-Thiouridines: Implications for Antisense Design and Targeting a Group I Intron
Abstract
Antisense compounds are designed to optimize selective hybridization of an exogenous oligonucleotide to a cellular target. Typically, Watson-Crick base pairing between the antisense compound and target provides the key recognition element. Uridine (U), however, not only stably base pairs with adenosine (A) but also with guanosine (G), thus reducing specificity. Studies of duplex formation by oligonucleotides with either an internal or a terminal 2- or 4-thiouridine (s(2)U or s(4)U) show that s(2)U can increase the stability of base pairing with A more than with G, while s(4)U can increase the stability of base pairing with G more than with A. The latter may be useful when binding can be enhanced by tertiary interactions with a s(4)U-G pair. To test the effects of s(2)U and s(4)U substitutions on tertiary interactions, binding to a group I intron ribozyme from mouse-derived Pneumocystis carinii was measured for the hexamers, r(AUGACU), r(AUGACs(2)U), and r(AUGACs(4)U), which mimic the 3' end of the 5' exon. The results suggest that at least one of the carbonyl groups of the 3' terminal U of r(AUGACU) is involved in tertiary interactions with the catalytic core of the ribozyme and/or thio groups change the orientation of a terminal U-G base pair. Thus thio substitutions may affect tertiary interactions. Studies of trans-splicing of 5' exon mimics to a truncated rRNA precursor, however, indicate that thio substitutions have negligible effects on overall reactivity. Therefore, modified bases can enhance the specificity of base pairing while retaining other activities and, thus, increase the specificity of antisense compounds targeting cellular RNA.
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2-thio-U forms a stronger base-pair with A than standard U by about 1 kcal/mol, while slightly destabilizing G:U pairing.
“A promising example is 2-thio-U (or 2-thio-T or other 5-substituted forms of 2-thio-U), based on thermodynamic data [34] that indicate that 2thio-U forms a stronger base-pair with A than does standard U (by about 1 kcal/mol), while slightly destabilizing G:U pairing.”
From The eightfold path to non-enzymatic RNA replication (2012) · cites this paper as [34]
The larger and more diffuse electron cloud of sulfur vs oxygen makes it more polarizable and may contribute to better stacking.
“In addition the larger and more diffuse electron cloud of sulfur vs oxygen makes it more polarizable and hence may contribute to better stacking.15,17”
From Replacing Uridine with 2-Thiouridine Enhances the Rate and Fidelity of Nonenzymatic RNA Primer Extension (2015) · cites this paper as [15]
Replacing U with s2U stabilizes base pairing with A while modestly destabilizing wobble pairing with G.
“We sought to address these challenges by replacing U with s2U, a nucleobase known to significantly stabilize base pairing with A while modestly destabilizing wobble pairing with G.14,15”
From Replacing Uridine with 2-Thiouridine Enhances the Rate and Fidelity of Nonenzymatic RNA Primer Extension (2015) · cites this paper as [15]
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- Authors
- Testa, S. M., Disney, M. D., Turner, D. H., & Kierzek, R.
- Affiliations
- Institute of Bioorganic Chemistry, Polish Academy of Sciences (PL)
- Corresponding author
- Turner, Douglas H.
- Venue
- Biochemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 1999-11-18
- Volume
- 38
- Issue
- 50
- Pages
- 16655-16662
- ISSN
- 0006-2960 (ISSN-L)
- 1520-4995
- OpenAlex
- W1989249011
- Cited by
- 122
- References
- 18
- Citations by year
- 2025: 6 · 2024: 4 · 2023: 4 · 2022: 7 · 2021: 6 · 2020: 5 · 2019: 9 · 2018: 5 · 2017: 5 · 2016: 5 · 2015: 5 · 2014: 5 · 2013: 6 · 2012: 6
- FWCI
- 0.58
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Biochemistry (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Institutes of Health (5r03ai045398-02)
- National Institute of Allergy and Infectious Diseases (AI45398)
- Fogarty International Center (1 R03 TW 1068-01, R03TW001068)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA and Nucleic Acid Chemistry
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Keywords
- 2-thiouridine, 4-thiouridine, RNA-RNA duplex, antisense oligonucleotides, Pneumocystis carinii, modified nucleosides, tertiary interactions, trans-splicing, 5' exon
- MeSH terms
- Animals, Binding Sites, Exons, Introns, Nucleic Acid Heteroduplexes, Pneumocystis, RNA Precursors, RNA, Ribosomal, Thermodynamics, Thiouridine, RNA, Catalytic, RNA, Antisense, Molecular Mimicry, Base Pairing, Mice
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