mRNA capping is a necessary post-transcriptional modification in eukaryotic cells that is involved in RNA stability, translation efficiency, and immune evasion. This involves inserting a modified guanine nucleotide at the 5' end of the incipient mRNA, creating a specific shape that is crucial for gene expression regulation. With the development of mRNA capping technology, new ways to maximise mRNA therapeutic effects have been developed, especially for the purpose of developing vaccines and gene therapy.
Figure 1. RNA cap structure: The cap consists of 7-methylguanosine (blue box) attached to the 5′ nucleoside of the messenger RNA strand via a 5′-5′ triphosphate bridge (pink box)[1].
Three enzymatic reactions typically participate in the cap:
Step 1: 5' Phosphate Removal
RNA triphosphatase (RTPase) removes one phosphate strand from the 5' triphosphate end of the nascent mRNA and leaves a diphosphate structure.
Step 2: Guanosine Addition
Guanylyltransferase (GTase) binds a guanine monophosphate (GMP) to the diphosphate tail through a 5'-5' triphosphate bridge, leaving the Cap0 structure (GpppN).
Step 3: Methylation
The guanosine is methylated at position 7 by RNA methyltransferase, and the resulting 7-methylguanosine (m7G) structure is formed. Still further changes (like 2'-O-methylation) can generate Cap1 or Cap2 structures, ensuring mRNA stability and immune permeability.
Such reactions are also likely to be co-transcribed with RNA polymerase II, and so cappping is closely associated with transcription lengthening.
Figure 2. Typical RNA capping mechanism[1].
| Protection from Degradation | The cap shields mRNA from exonuclease degradation and keeps it intact in the cytoplasm. |
| Facilitation of Translation | The cap also calls upon eukaryotic translation initiation factors (eIFs) that direct the ribosome to the mRNA, which promotes the production of proteins. |
| Promotion of Nuclear Export | Capping binds to the nuclear export machinery and helps mRNA get from the nucleus to the cytoplasm. |
| Splicing and Polyadenylation Coordination | This capping happens in close association with splicing and polyadenylation so that mRNA maturation coincides. |
| Immune Evasion | Good capping keeps mRNA from being detected by pattern recognition receptors (PRRs) (e.g., RIG-I), which lowers innate immune response. This property is particularly useful when designing mRNA-based drugs. |
Co-transcriptional capping integrates the capping process with transcription. During in vitro transcription (IVT) using an RNA polymerase, a cap analog (e.g., m7GpppG) is integrated into the nascent RNA.
This has the advantage of being efficient and simple and can be quickly integrated into the IVT workflow. However, it has the potential for reverse capping, in which cap analogs are integrated in the wrong orientation, thereby reducing translation efficiency.
Post-transcriptional capping involves the enzymatic addition of cap structures to pre-synthesized RNA. Key enzymes include:
(1) Varroa Capping Enzyme (VCE): Adds the Cap0 structure.
(2) 2'-O-methyltransferase: converts Cap0 to Cap1, enhancing stability and translation.
Post-transcriptional capping has near-complete capping efficiency and the flexibility to produce highly modified cap structures. However, additional processing steps are required.
CleanCap is a new type of cotranscriptional cap that has no need for traditional cap analogs. It directly returns Cap1 structures with very little by-product. CleanCap avoids reverse caps and makes translational efficiency in the workflow easy.
CleanCap has been tested in the manufacture of mRNA vaccines (e.g., Moderna's mRNA-1273), so it's of clinical utility.
Figure 3. Eukaryotic cap structures and cap analogs: (A) Eukaryotic cap structures. (B) Structures of antiretroviral cap analogs used in standard cotranscriptional capsidization. (C) Structure of the CleanCap AG Cap1 trimer. (D) Proposed mechanism of CleanCap cotranscription initiation[3].
Innovation in capping technologies is always ongoing to scale production, lower prices, and increase therapeutic value. Chemical modification of cap architecture and combination with synthetic biology approaches are promising research. Alfa Chemistry will continue to provide premium reagents and technical support to drive mRNA capping techniques for science and clinical use.
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