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Abstract Inosine is an important RNA modification, furthermore RNA oxidation has gained interest due, in part, to its potential role in the development/progression of disease as well as on its impact on RNA structure and function. In this report we established the base pairing abilities of purine nucleobases G, I, A, as well as their corresponding, 8‐oxo‐7,8‐dihydropurine (common products of oxidation at the C8‐position of purines), and 8‐bromopurine (as probes to explore conformational changes), derivatives, namely 8‐oxoG, 8‐oxoI, 8‐oxoA, 8‐BrG, and 8‐BrI. Dodecamers of RNA were obtained using standard phosphoramidite chemistry via solid‐phase synthesis, and used as models to establish the impact that each of these nucleobases have on the thermal stability of duplexes, when base pairing to canonical and noncanonical nucleobases. Thermal stabilities were obtained from thermal denaturation transition (Tm) measurements, via circular dichroism (CD). The results were then rationalized using models of base pairs between two monomers, via density functional theory (DFT), that allowed us to better understand potential contributions from H‐bonding patterns arising from distinct conformations. Overall, some of the important results indicate that: (a) an anti‐I:syn‐A base pair provides thermal stability, due to the absence of the exocyclic amine; (b) 8‐oxoG base pairs like U, and does not induce destabilization within the duplex when compared to the pyrimidine ring; (c) a U:G wobble‐pair is only stabilized by G; and (d) 8‐oxoA displays an inherited base pairing promiscuity in this sequence context. Gaining a better understanding of how this oxidatively generated lesions potentially base pair with other nucleobases will be useful to predict various biological outcomes, as well as in the design of biomaterials and/or nucleotide derivatives with biological potential.
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Base pairing, structural and functional insights into N4-methylcytidine (m4C) and N4,N4-dimethylcytidine (m42C) modified RNA
Abstract The N4-methylation of cytidine (m4C and m42C) in RNA plays important roles in both bacterial and eukaryotic cells. In this work, we synthesized a series of m4C and m42C modified RNA oligonucleotides, conducted their base pairing and bioactivity studies, and solved three new crystal structures of the RNA duplexes containing these two modifications. Our thermostability and X-ray crystallography studies, together with the molecular dynamic simulation studies, demonstrated that m4C retains a regular C:G base pairing pattern in RNA duplex and has a relatively small effect on its base pairing stability and specificity. By contrast, the m42C modification disrupts the C:G pair and significantly decreases the duplex stability through a conformational shift of native Watson-Crick pair to a wobble-like pattern with the formation of two hydrogen bonds. This double-methylated m42C also results in the loss of base pairing discrimination between C:G and other mismatched pairs like C:A, C:T and C:C. The biochemical investigation of these two modified residues in the reverse transcription model shows that both mono- or di-methylated cytosine bases could specify the C:T pair and induce the G to T mutation using HIV-1 RT. In the presence of other reverse transcriptases with higher fidelity like AMV-RT, the methylation could either retain the normal nucleotide incorporation or completely inhibit the DNA synthesis. These results indicate the methylation at N4-position of cytidine is a molecular mechanism to fine tune base pairing specificity and affect the coding efficiency and fidelity during gene replication.
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- PAR ID:
- 10223807
- Date Published:
- Journal Name:
- Nucleic Acids Research
- Volume:
- 48
- Issue:
- 18
- ISSN:
- 0305-1048
- Page Range / eLocation ID:
- 10087 to 10100
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1093/nar/gkaa737
