Combining Chemical Synthesis and Enzymatic Methylation to Access Short RNAs with Various 5′ Caps

Abstract

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 7mG cap is hypermethylated at the N2‐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.

Photocaged 5′ cap analogues for optical control of mRNA translation in cells

“…The enzymes 5'-methylthioadenosine nucleosidase (MTAN), LuxS, hDcpS H277N, eIF4E and hTgs were produced and purified as previously described 16 , 35 , 49 , 50 .…”

Quantification of mRNA cap-modifications by means of LC-QqQ-MS

“…Adaptations of this method should also be suitable to quantify different enzymatic modifications in the mRNA cap, i.e. methylation or functionalization of the terminal N 2 -position or the 2′- O -position of the TSN [ 51 – 55 ]. We therefore anticipate broad applicability of this protocol to investigating MTase-catalyzed mRNA cap modifications.…”

Chemical Synthesis of Modified RNA

“…For academic research applications, a reasonable compromise for RNA synthesis is provided with ribose 2'-O-TBDMS (tert-butyldimethylsilyl) [10] or 2'-O-TOM [(triisopropylsilyl)oxy]methyl [11] (Figure 2a) in combination with nucleobase acetyl-, phenoxyacetyl-, or amidine protections (Figure 2b), followed by a 2-step cleavage/release protocol of the fully assembled RNAs using AMA (ammonium hydroxide/aqueous methylamine), and then, tetra-n-butylammonium fluoride (TBAF) or triethylamine trihydrofluoride (TEA 3HF), both at elevated temperatures. [9] Over the years, many reports on alternative 2'-OH protection concepts testify to the continuing search for superior protective groups (Figure 2c), which has manifested itself in the imino-2-propanoate, [12] propionyloxymethyl (PrOM), [13] {[2,2-dimethyl-2-(2-nitrophenyl)acetyl]oxy} methyl (DAM), [14] 2-cyano-2,2-dimethylethanimine-Noxymethyl, [15] 1,1-dioxo-λ 6 -thio-morpholine-4-carbothioate (TC, thionocarbamate), [16] pivaloyloxymethyl (PivOM), [17] (2-cyanoethoxymethyl) (CEM), [18] [(2-nitrobenzyl)oxy]methyl] (nbm), [19] [(S)-1-(2-nitrophenyl)ethoxy]methyl ((S)npeom), [20] and bis(acetoxyethoxy)methyl (ACE) [21] groups (Figure 2d). Several review articles cover some of the above mentioned and other 2'-O protection strategies.…”