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Combining Chemical Synthesis and Enzymatic Methylation to Access Short RNAs with Various 5′ Caps
Author(s) -
Muthmann Nils,
Guez Théo,
Vasseur JeanJacques,
Jaffrey Samie R.,
Debart Françoise,
Rentmeister Andrea
Publication year - 2019
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201900037
Subject(s) - small nucleolar rna , methyltransferase , rna , methylation , biology , transcription (linguistics) , non coding rna , long non coding rna , biochemistry , computational biology , genetics , dna , gene , linguistics , philosophy
Eukaryotic RNAs are heavily processed, including co‐ and post‐transcriptional formation of various 5′ caps. In small nuclear RNAs (snRNAs) or small nucleolar RNAs (snoRNAs), the canonical 7m G cap is hypermethylated at the N 2 ‐position, whereas in higher eukaryotes and viruses 2′‐O‐methylation of the first transcribed nucleotide yields the cap1 structure. The function and potential dynamics of several RNA cap modifications have not been fully elucidated, which necessitates preparative access to these caps. However, the introduction of these modifications during chemical solid‐phase synthesis is challenging and enzymatic production of defined short and uniform RNAs also faces difficulties. In this work, the chemical synthesis of RNA is combined with site‐specific enzymatic methylation by using the methyltransferases human trimethylguanosine synthase 1 (hTgs1), trimethylguanosine synthase from Giardia lamblia (GlaTgs2), and cap methyltransferase 1 (CMTR1). It is shown that RNAs with di‐and trimethylated caps, as well as RNAs with caps methylated at the 2′‐O‐position of the first transcribed nucleotide, can be conveniently prepared. These highly modified RNAs, with a defined and uniform sequence, are hard to access by in vitro transcription or chemical synthesis alone.