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.

Calculations of pKa Values for a Series of Naturally Occurring Modified Nucleobases

“…In one report of duplex RNAs containing m4C, there was a slight decrease in duplex stability when the modification was present. 108 This decrease in stability for G-m4C correlated with an observed increased ΔpK a compared to G−C. In contrast, other studies showed an opposite correlation between oligonucleotide stabilities and ΔpK a between donor and acceptor pairs.…”

“…We used the Δp K a rule with adjusted theoretical p K a values and compared to published stability data. In one report of duplex RNAs containing m4C, there was a slight decrease in duplex stability when the modification was present . This decrease in stability for G-m4C correlated with an observed increased Δp K a compared to G–C.…”

Challenges with Simulating Modified RNA: Insights into Role and Reciprocity of Experimental and Computational Approaches

“… ( a ) An example of how a modification can affect base-pairing interactions. Here, N 4 , N 4 -dimethylcytidine (in pink) has only two possible base-pairing sites on its W-C-F edge due to the double substitution of methyl groups on the amine, while a typical G: C base pair would have three [ 36 ]. ( b ) An example of three base pairs, one only with canonical bases, and the other with one RNA modification (5-methylcytidine, illustrated in pink).…”

The Research of G–Motif Construction and Chirality in Deoxyguanosine Monophosphate Nucleotide Complexes

“…As we know, the visualization of these “host–guest” interactions based on X-ray crystallography will help understand the details of interactions between functional intercalations and targets, as well as the nucleotide conformational polymorphism changes. Although some related researches have been reported by single-crystal XRD ( Monestier et al, 2017 ; Chu et al, 2019 ; Geng et al, 2019 ; Satange et al, 2019 ; Mao et al, 2020 ), the well-defined structure of “host–guest” interaction about intercalator-mismatched DNA remains as a fundamental and challenging issue for comprehensive understanding of the biological processes in genomic DNA. A proper design and synthesis of molecular building blocks is an effective methodological strategy to obtain innovative functional materials, which is an ultimate target in supramolecular chemistry.…”