3-Methylcytidine is a modified nucleoside derived from cytidine, where a methyl group is attached to the third carbon of the cytidine molecule. This modification can occur naturally in various RNA molecules, playing a role in the regulation of gene expression and the stability of RNA. Modified nucleosides like 3-methylcytidine are often found in tRNA, rRNA, and other non-coding RNAs, contributing to the proper functioning of the cellular machinery.
Reference Reading
1. Roles and dynamics of 3-methylcytidine in cellular RNAs
Katherine E Bohnsack, Nicole Kleiber, Nicolas Lemus-Diaz, Markus T Bohnsack. Trends Biochem Sci. 2022 Jul;47(7):596-608. doi: 10.1016/j.tibs.2022.03.004.
Modified nucleotides within cellular RNAs significantly influence their biogenesis, stability, and function. As reviewed here, 3-methylcytidine (m3C) has recently come to the fore through the identification of the methyltransferases responsible for installing m3C32 in human tRNAs. Mechanistic details of how m3C32 methyltransferases recognize their substrate tRNAs have been uncovered and the biogenetic and functional relevance of interconnections between m3C32 and modified adenosines at position 37 highlighted. Functional insights into the role of m3C32 modifications indicate that they influence tRNA structure and, consistently, lack of m3C32 modifications impairs translation. Development of quantitative, transcriptome-wide m3C mapping approaches and the discovery of an m3C demethylase reveal m3C to be dynamic, raising the possibility that it contributes to fine-tuning gene expression in different conditions.
2. Structure-Activity Relationship of 3-Methylcytidine-5'-α,β-methylenediphosphates as CD73 Inhibitors
Mirko Scortichini, Riham Mohammed Idris, Susanne Moschütz, Antje Keim, Veronica Salmaso, Clemens Dobelmann, Paola Oliva, Karolina Losenkova, Heikki Irjala, Samuli Vaittinen, Jouko Sandholm, Gennady G Yegutkin, Norbert Sträter, Anna Junker, Christa E Müller, Kenneth A Jacobson. J Med Chem. 2022 Feb 10;65(3):2409-2433. doi: 10.1021/acs.jmedchem.1c01852.
We recently reported N4-substituted 3-methylcytidine-5'-α,β-methylenediphosphates as CD73 inhibitors, potentially useful in cancer immunotherapy. We now expand the structure-activity relationship of pyrimidine nucleotides as human CD73 inhibitors. 4-Chloro (MRS4598 16; Ki = 0.673 nM) and 4-iodo (MRS4620 18; Ki = 0.436 nM) substitution of the N4-benzyloxy group decreased Ki by ~20-fold. Primary alkylamine derivatives coupled through a p-amido group with a varying methylene chain length (24 and 25) were functionalized congeners, for subsequent conjugation to carrier or reporter moieties. X-ray structures of hCD73 with two inhibitors indicated a ribose ring conformational adaptation, and the benzyloxyimino group (E configuration) binds to the same region (between the C-terminal and N-terminal domains) as N4-benzyl groups in adenine inhibitors. Molecular dynamics identified stabilizing interactions and predicted conformational diversity. Thus, by N4-benzyloxy substitution, we have greatly enhanced the inhibitory potency and added functionality enabling molecular probes. Their potential as anticancer drugs was confirmed by blocking CD73 activity in tumor tissues in situ.
3. Synthesis and Purification of N3 -Methylcytidine (m3 C) Modified RNA Oligonucleotides
Johnsi Mathivanan, Jinxi Du, Song Mao, Ya Ying Zheng, Jia Sheng. Curr Protoc. 2021 Nov;1(11):e307. doi: 10.1002/cpz1.307.
This protocol describes a step-by-step chemical synthesis approach to prepare N3 -methylcytidine (m3 C) and its phosphoramidite. The method for synthesizing m3 C starts from commercially available cytidine, and proceeds via N3 -methylation in the presence of MeI, which generates the N3 -methylcytidine (m3 C) nucleoside, followed by the installation of several protecting groups at sites that include the 5'-hydroxyl group (4,4'-dimethoxytrityl protection), the 4-amino group (benzoyl protection), and the 2'-hydroxyl group (tert-butyldimethylsilyl, TBDMS, protection). Standard phosphoramidite chemistry is applied to prepare the final m3 C phosphoramidite for solid-phase synthesis of a series of RNA oligonucleotides. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of N3 -methylcytidine (m3 C) and its phosphoramidite Basic Protocol 2: Automated synthesis of m3 C modified RNA oligonucleotides.Keywords:N3-methylcytidine; RNA modification; phosphoramidite; solid-phase synthesis.
