Synthesis of N4 -Methylcytidine (m4 C) and N4 ,N4 -Dimethylcytidine (m4 2 C) Modified RNA

Ya Ying Zheng; Song Mao; Jia Sheng

doi:10.1002/cpz1.248

Synthesis of N⁴ -Methylcytidine (m⁴ C) and N⁴ ,N⁴ -Dimethylcytidine (m⁴ ₂ C) Modified RNA

Curr Protoc. 2021 Sep;1(9):e248. doi: 10.1002/cpz1.248.

Authors

Ya Ying Zheng¹, Song Mao¹, Jia Sheng¹

Affiliation

¹ Department of Chemistry and The RNA Institute, University at Albany, State University of New York, Albany, New York.

PMID: 34529364
DOI: 10.1002/cpz1.248

Abstract

This article summarizes the protocols for phosphoramidite chemistry and solid phase synthesis of RNA oligonucleotides containing N⁴ -methylcytidine (m⁴ C) and N⁴ ,N⁴ -dimethylcytidine (m⁴ ₂ C) residues for base-pairing, structural, and enzymatic activity studies. The two key m⁴ C and m⁴ ₂ C phosphoramidite building blocks can be synthesized starting from the partially protected cytidine nucleosides, followed by solid-phase synthesis and HPLC purification of the modified target RNA oligonucleotides. These modified RNA strands are then prepared for base pairing stability, specificity, and structural studies using UV-melting temperature (T_m ) measurements and X-ray crystallography. Functional studies are performed by reverse transcription assays in primer extension reactions employing different enzymes. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Chemical synthesis of m⁴ C phosphoramidite Basic Protocol 2: Synthesis of m⁴ ₂ C phosphoramidite Basic Protocol 3: Synthesis and purification of m⁴ C and m⁴ ₂ C containing RNA oligonucleotides.

Keywords: N4,N4-dimethylcytidine (m42C); N4-methylcytidine (m4C); X-ray crystallography; base pairing; nucleic acids modification; thermostability.

MeSH terms

Base Pairing
Cytidine*
Nucleosides
Oligonucleotides
RNA*

Substances

Nucleosides
Oligonucleotides
Cytidine
RNA