Glen Report 35-21: CleanCap® M6

Author:  Chunping Xu, Ph.D.,
TriLink BioTechnologies

Introduction:

The m7G (N7-Methyl guanosine) cap structure is present in all eukaryotic mRNAs and plays an essential role in the cap-dependent initiation of protein synthesis. Another role of the mRNA cap is to stabilize mRNA and prevent it from 5′ to 3′ exonuclease cleavage. Various dinucleotide and trinucleotide cap analogs have been reported in the literature so far for co-transcriptional mRNA capping.^1,2 Compared to dinucleotide cap analogs such as ARCA and related derivatives, TriLink’s CleanCap AG (m7GpppAG) can enable more efficient mRNA capping since T7 RNA polymerase initiates mRNA synthesis  on CleanCap AG by hybridizing two nucleotides (AG)  to the DNA template. In contrast, ARCA initiates mRNA synthesis by binding a single nucleotide (G) to the DNA template, and there is competition from GTP, which will result in lower capping efficiency (Figure 1).

Figure 1. ARCA versus CleanCap reagent

N6-Methyl adenosine (^m6A) is the most abundant internal modification in mammalian mRNA and modulates numerous cellular processes such as nuclear export, mRNA splicing, polyadenylation, stability and translation. N6, 2′-O-Dimethyl adenosine (^m6A_m) is an extension of the 5′ cap that can occur on the first transcribed nucleotide if it is an adenosine. The addition of ^m6A_m to 5′-UTR has been shown to play a role in both mRNA translation and stability. Even though most translation is achieved through recognition of the 5′ cap, for select mRNA,  the ^m6A modification has been shown to facilitate  cap-independent translation during stress.^3-6

CleanCap M6 development: 

In our efforts to find more efficient cap analogs, we wanted to introduce the ^m6A_m modification into CleanCap AG and study the effect of the ^m6A_m on mRNA translation. Using our standard mRNA in vitro transcription (IVT) conditions, this cap results in low yield and low capping efficiency. As a result, new IVT conditions had to be developed.⁷

Figure 2. CleanCap AG Analogs

CleanCap AG 3′OMe and CleanCap M6 (Figure 2) have been incorporated into Wasabi mRNA (modified GFP) and tested in a cell-based assay for protein expression in HeLa cells. Significant enhancement in protein expression was observed. Protein expression of mWasabi by in vitro translation was increased by more than two-fold when CleanCap M6 was utilized, relative to that of CleanCap AG 3′-OMe (Figure 3). The three versions of CleanCap analogs (CleanCap AG, CleanCap AG 3′OMe and CleanCap M6, Figure 2) were also evaluated in vivo.  When incorporated into FLuc mRNA and tested in mice for protein expression, we observed a significant increase in protein translation going from CleanCap AG  to CleanCap AG 3′OMe and also from CleanCap AG 3′OMe to CleanCap M6 (Figures 4A and 4B). CleanCap M6 incorporated into other mRNA constructs, such as EPO, also show enhancement in protein expression. 

CleanCap M6 has been shown to give the best protein expression in all the cap analogs we have tested so far (Figures 4A and 4B). The impact of m6Am on protein translation has been reported in the literature by other groups.^3-6 Although, the exact mechanism is not clear yet, we believe the ^m6A_m nucleotide hinders the decapping process and stabilizes mRNA in cells, which will lead to protein translation improvement. 

Figure 3: CleanCap M6 modification promotes in vitro protein translation (mWasabi). All groups are significantly different, technical replicates *** p < 0.001, t-tailed T-test. Error bars are standard error of mean. n = 5/group. HeLa cells were transfected with mRNA coding mWasabi, capped with either CleanCap AG 3′OMe or CleanCap M6, and fluorescence was measured at 24 hours.

Figure 4: CleanCap M6 mRNA promotes in vivo Luciferase translation in mice. Performance of FLuc mRNA in an LNP-formulated, tail vein delivered mouse model, 1 mg/kg dose per group. Luciferase activity, as photons per second, was measured after luciferin injection. The difference between the groups was cap analog structure only. All other variables were controlled. A. All groups are significantly different, ** p < 0.01, one-way ANOVA. Error bars are standard error of mean. n = 5/group. B. Whole body luminescence.

Applications: 

With TriLink’s streamlined mRNA co-transcriptional capping process, CleanCap M6 can be widely used in mRNA capping to facilitate mRNA vaccine or therapeutic development. After we launched this new product in May, many customers have already tested it. CleanCap M6 can be used to replace the current enzymatic or ARCA capping technology due to the benefits associated with CleanCap M6:

1. Enhanced protein expression
2. High capping efficiency
3. Low levels of double-stranded RNA
4. Faster turn-around time compared to both ARCA  and enzymatic capping
5. Lower capping cost at a larger scale compared to enzymatic cappin

For therapeutic development, due to the higher potency, CleanCap M6 capped mRNA can be used at a lower dose to minimize an immune response.

References:

1. Muttach, F., et al. (2017). “Synthetic mRNA capping.” Beilstein J Org Chem 13: 2819-2832.
2. Kowalska, J., et al. (2008). “Synthesis and characterization of mRNA cap analogs containing phosphorothioate substitutions that bind tightly to eIF4E and are resistant to the decapping pyrophosphatase DcpS.” RNA 14(6): 1119-1131.
3. Mauer, J., et al. (2017). “Reversible methylation of m(6)A(m) in the 5’ cap controls mRNA stability.” Nature 541(7637): 371-375.
4. Liu, A. and X. Wang (2022). “The Pivotal Role of Chemical Modifications in mRNA Therapeutics.”  Front Cell Dev Biol 10: 901510.
5. Catacalos, C., et al. (2022). “Epitranscriptomics in parasitic protists: Role of RNA chemical modifications in posttranscriptional gene regulation.” PLoS Pathog 18(12): e1010972.
6. Sikorski, P. J., et al. (2020). “The identity and methylation status of the first transcribed nucleotide in eukaryotic mRNA 5’ cap modulates protein expression in living cells.” Nucleic Acids Res  48(4): 1607-1626.
7. CleanCap Reagent M6-(N-7453), https://www.trilinkbiotech.com/cleancap-reagent-m6.html.