Department of Chemistry and Pharmacy, Institute for Biochemistry, University of Münster, Wilhelm-Klemm-Strasse 2, 48149, Münster, Germany.
Institut des Biomolécules Max Mousseron (IBMM), Université de Montpellier, CNRS, ENSCM, Campus Triolet UM, Place Eugène Bataillon, 34095, Montpellier, France.
Chembiochem. 2019 Jul 1;20(13):1693-1700. doi: 10.1002/cbic.201900037. Epub 2019 May 27.
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 G cap is hypermethylated at the N -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.
真核 RNA 经历了广泛的加工，包括转录和转录后形成各种 5' 帽结构。在小核 RNA（snRNA）或小核仁 RNA（snoRNA）中，典型的 G 帽在 N-位上高度甲基化，而在高等真核生物和病毒中，第一个转录核苷酸的 2'-O-甲基化生成帽 1 结构。几种 RNA 帽修饰的功能和潜在动力学尚未完全阐明，这需要制备这些帽结构。然而，在化学固相合成中引入这些修饰具有挑战性，并且酶法生产具有明确结构的短链且均一的 RNA 也面临困难。在这项工作中，通过使用人三甲基鸟苷合酶 1（hTgs1）、蓝氏贾第鞭毛虫三甲基鸟苷合酶（GlaTgs2）和帽甲基转移酶 1（CMTR1）进行特异性酶促甲基化，将 RNA 的化学合成与位点特异性酶促甲基化相结合。结果表明，可方便地制备二甲基化和三甲基化帽 RNA，以及 2'-O-位被甲基化的第一个转录核苷酸帽 RNA。这些高度修饰的 RNA 具有明确且均一的序列，很难通过体外转录或单独的化学合成获得。
