mRNA Capping Method

Summary

Mature mRNAs require 5' cap structures, which are evolutionarily conserved modifications in eukaryotes and are essential for the translation of most proteins in vivo. The m7G cap structure consists of 7-methyl guanosine (m7G) triphosphate linked to the 5' end of the mRNA via a 5'-5' triphosphate bridge (m7G cap), that is the so-called cap 0 structure. The RNA with the 2'-O methylation at the first nucleotide (cap1), is a modification that is commonly observed in eukaryotes, while the additional methyl group on the second nucleotide is called cap2. Cap1 structure can improve the stability of mRNA and enhance its expression ability in cell transfection and microinjection experiments.

Vaccinia Capping System (Based on the Vaccinia Virus Capping Enzyme)

This approach is based on post-transcriptional capping. The capping enzyme of the vaccinia virus is a complex of the two viral proteins D1(catalytic activity) and D12(no catalytic activity but stabilizes the D1 protein). The D1 protein contains three activities:

• First, the triphosphatase activity hydrolyzes the RNA 5' triphosphate into a 5' diphosphate and inorganic phosphate.
• Second, the guanylyltransferase activity transfers GMP from GTP onto the 5' end of the diphosphate RNA. In this step, the 5'-5' triphosphate bond is formed and pyrophosphate is released.
• Finally, the methyltransferase activity uses SAM (S-adenosyl methionine) as a methyl donor to methylate the guanine N7 position. In addition, the vaccinia virus also contains a 2'-o-methyltransferase (VP39) that converts the Cap0 structure to Cap1 structure.

Vaccinia Capping System can be used to produce capped mRNAs (Cap0 and Cap1) for large-scale structural studies. This method is universally applicable with cost-efficient advantage, of which, the efficiency of the cap-capping reaction is largely independent of the sequence, length, and secondary structure of mRNA. The produced capped mRNAs can be directly used for quantitative biophysical studies including Fluorescence anisotropy and high-resolution NMR spectroscopy.

Fig. 1 Schematic representation of enzymatic 5'-cap formation in eukaryotic mRNAs. (Muttach F, 2017)

Co-transcriptional Capping Method In Vitro Transcription (IVT)

Through the co-transcriptional capping approach, cap analogs (m7GpppG, m7GpppA, GpppA, GpppG) can be directly added into the IVT of a DNA template, and they are incorporated at the 5' end by RNA polymerases with relaxed substrate specificity (e.g., T3, T7, or SP6 RNA polymerases) to eventually produce their respective 5'-capped mRNAs. Internal incorporation of cap analogs during IVT does not occur, because cap analogs lack a free 5'-triphosphate. Of note, when m7GpppG is used as a cap analog, miscapped RNA with reverse-orientation cap analogs is produced in addition to correctly capped RNA. However, such a wrong phenomenon with false extension at 3'-OH could be effectively prevented by the incorporation of an anti-reverse cap analog (ARCA).

Fig. 2 Schematic representation of co-transcriptional capping with different cap analogs. (Muttach F, 2017)

Chemical Synthesis of Capped mRNA

Chemical synthesis of capped mRNA is based on a solid-phase synthesis of RNA coupled with chemical or enzymatic installation of 5'-caps.

A 2,2,7-trimethylguanosine (TMG)-capped RNA was synthesized starting from an RNA tetramer which was subjected to 5'-terminal pyrophosphorylation followed by a reaction with a 2,2,7-trimethylguanosine 5'-phosphorimidazolide derivative. Subsequent cleavage from the solid support was achieved using 80% AcOH (at room temperature, 24 h) and TBDMS protecting groups were removed with HCl (pH2, at room temperature, 12 h).

Large-scale production of mRNAs with cap0 or cap1 through the combination of solid-phase synthesis and enzymatic modification. To avoid the dilemma of m7G instability, unmethylated capped mRNA was first synthesized using the phosphoramidite 2'-O-pivaloyloxymethyl method, followed by enzymatic N7 methylation using human (guanine-N7) -methyltransferase. The cap1 structure can also be obtained through 2'-OH methylation of the terminal nucleotide. Deprotection conditions: DBU (1,8-diazadicyclo[5,4,0]undec-7-ene) in acetonitrile (at room temperature, 3 min) followed by treatment with aqueous ammonia (at room temperature, 3 h).

Solid-phase synthesis provides the flexibility to introduce modified nucleotides at specific positions and yields the capped mRNAs independent of the sequence.

Fig. 3 Synthesis of cap-containing mRNA by solid-phase synthesis. (Muttach F, 2017)

Fig. 4 The General course of the synthesis of 5′-7mGppp-mRNAs. (Thillier Y, 2012)

Combining Chemical and Enzymatic Methods: Primer Extension

Replicative DNA polymerase from Thermococcus gorgonarius (Tgo) was engineered into a DNA-dependent RNA polymerase (called TGK) capable of producing RNA up to 1,700 nt long from ssDNA templates and RNA primers. In contrast to most other RNA polymerases used for conventional in vitro transcription, primer-dependent RNA synthesis does not require the use of pppG to initiate RNA synthesis. As a result, TGK accepts many variants at the 5'-end, including oligonucleotide primers containing the desired cap. This approach combines the flexibility of RNA synthesis with the continuous synthesis capability of RNA polymerases for the preparation of long and cap-modified mRNAs. Through this system, diverse biologically relevant RNAs such as GFP RNA, firefly luciferase RNA, and m7Gpppm6Am-RNA can be generated.

Click Chemistry for the Preparation Of Capped RNAs and Cap Analogs

Different hypermethylated cap analogs with a 2'-azide moiety allow a reaction with alkyne-modified RNA to produce cap-modified RNA in the CuAAC reaction. This capping route also applies to alkyne-modified triphosphorylated RNAs and 5'-azide-modified methylguanosine, resulting in capped RNAs containing a triazole bond after the CuAAC reaction. In a similar method, a 5'-azido-modified RNA was prepared via solid-phase synthesis and reacted with an alkyne-functionalized m7G-cap analog in a CuAAC reaction.