期刊
CHEMBIOCHEM
卷 20, 期 13, 页码 1693-1700出版社
WILEY-V C H VERLAG GMBH
DOI: 10.1002/cbic.201900037
关键词
methylation; RNA; solid-phase synthesis; synthesis design; transferases
资金
- NIH [R01CA186702]
- DFG [RE2796/2-1, RE2796/6-1]
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.
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