The Cap structure is an essential component in the development of mRNA vaccines and therapeutics. Synthetic mRNA technology relies on Cap1 to enhance stability, translational efficiency, and reduce the innate immune recognition of mRNA by toll-like receptors (TLRs) and other immune sensors, minimizing unwanted immune activation. This prevents undesired inflammatory responses, thereby improving the stability and effectiveness of the therapeutic mRNA in human cells.
Early Discovery and Cap0 Structure
In the mid-1970s, research into eukaryotic mRNA uncovered a 5' terminal structure now known as Cap0, where an N7-methylguanosine cap (m7G) is attached to the first nucleotide at the 5'-end. This modification was found to protect mRNA from degradation by exonucleases, assist in nuclear export, and promote ribosome recognition for translation initiation. Cap0 was the first characterized cap structure, with its methylation limited to the guanosine cap itself. The discovery of this 5'-cap and its functions marked a significant breakthrough in understanding eukaryotic mRNA caps and their role in mRNA modifications.
The Emergence of Cap1
The Cap1 structure, a critical feature of eukaryotic mRNA, plays a pivotal role in mRNA stability, translation, and immune recognition. The mRNA cap structure, consisting of an N7-methylguanosine (m7G) linked to the first nucleotide via a 5'-5' triphosphate linkage, evolved from early studies on eukaryotic mRNA post-transcriptional modifications in the 1970s.
Structure of 5′-end capped mRNAs.【1】 Further investigations revealed that in most eukaryotic mRNAs, the first nucleotide following the cap (position +1) is also methylated at the 2'-O position of the ribose, a process known as 2'-O-methylation. This discovery, known as the Cap1 structure (m7GpppNm), added another layer of functional significance to mRNA modifications. The Cap1 modification was found to fine-tune mRNA stability and prevent immune recognition by the innate immune system, particularly in mammalian cells, where it helps evade recognition by pattern recognition receptors like RIG-I, TLRs, and MDA5【2】. This was crucial for both mRNA metabolism and the advancement of mRNA-based technologies, including mRNA vaccination and gene therapy applications.
Technical Challenges in mRNA Industry and Current Solutions
Several technical challenges remain in the widespread application and optimization of mRNA vaccines, including issues with high-dose mRNA, immune responses, stability, and delivery. A significant challenge in the mRNA industry is the need for high doses of mRNA to elicit a robust immune response. This is due to several factors:
Rapid Degradation: mRNA is inherently unstable and susceptible to degradation by decapping enzymes and ribonucleases (RNases) present in biological systems.
Limited Translational Efficiency: The efficiency with which mRNA is translated into protein can vary, requiring higher amounts of mRNA to generate sufficient antigen levels for an immune response. The translation efficiency is related to the affinity of Cap1 and translation initiation factor eIF4E.
The formation of the basic translation initiation complex in eukaryotes.【3】
Immunogenicity and Unwanted Immune Reactions: The third biggest hurdle is the innate immune response that can be triggered by the mRNA itself, especially for dsRNA or incomplete mRNA. While a certain level of immune activation is desired to stimulate the adaptive immune system, excessive activation can lead to inflammatory responses, which may reduce the effectiveness of the vaccine or cause side effects.
Capping Technology History
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Enzymatic Capping Technology: Enzymatic capping, introduced in the early days of mRNA research, involves using capping enzymes like guanylyltransferase and methyltransferase to add a natural 5' cap post-transcriptionally. This method ensures high fidelity and capping efficiency in mRNA translation, particularly for therapeutic applications.
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Synthetic Cap Analogues (1980s-2000s): Researchers developed synthetic cap analogs for in vitro synthesis of capped mRNA, helping to study mRNA function and gene expression. Anti-Reverse Cap Analog (ARCA) is a synthetic cap analog designed to prevent incorrect cap incorporation during mRNA synthesis, improving the translation efficiency of mRNA for vaccines and gene therapies. However, the capping efficiency and yield of ARCA capping is low.
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Cap1 Technology (2010s): TriLink's Cap1 is a co-transcriptional capping method that simplifies the process by incorporating the cap directly during mRNA synthesis. This method enhances efficiency and yields a high percentage of correctly capped mRNA, making it ideal for large-scale therapeutic applications like mRNA vaccines.
Currently, enzymatic capping technologies have been critical in the development of mRNA-based therapies, including COVID-19 vaccines, ensuring the stability and effective translation of therapeutic mRNA.
Comparision of Enzymatic capping, the next generation LZCap Capping and the first generation Capping (ARCA) method
Developing Next Generation Cap Analogs
We aimed to develop cap analogs with resistance to decapping enzymes, high affinity to eIF4E to improve translation efficiency, and lower immunogenicity. These advancements are crucial for enhancing the effectiveness of mRNA-based therapies, including anti-cancer treatments and gene therapy applications.
Designing LZCap
In designing LZCap, we mainly focused on creating a cap analog with high affinity to eIF4E. Enzymes have a relatively "specific" recognition of substrates. Therefore, when designing a new cap structure, we strive to maintain similarity with natural/known structures as much as possible. The natural structure has a ribose 3' OH, which can be modified (e.g., methylation). Based on this consideration, we chose to add a carbon at the 3' position for novelty, followed by an NH to mimic the hydrogen bonding of OH, and then an acetyl group to reduce the basicity of NH and enhance its hydrogen bonding capability. The activity of LZCap is better than methylated natural cap, possibly due to increased hydrogen bonding. Compared to the methyl and methoxy groups, the acetyl amino group may also increase van der Waals interactions between the substrate (cap) and the initiating factor (enzyme).
Stability of the Acetyl Amino Group
The acetyl amino group is already sufficiently stable. It is much more stable than the 7-methylated position and the phosphodiester bond, which are the least stable parts of the cap.
Yield and Capping Efficiency of LZCap
With LZCap AG(3'Acm) cap, the luciferase mRNA capping efficiency is about 97.59%, and up to 200 μg capped mRNA can be obtained with 1 μg DNA template in a standard in vitro transcription (IVT) reaction with T7 RNA polymerase. The mRNA purity is up to 99% after simple LiCl precipitation. This high capping efficiency and yield make LZCap an attractive option for various applications, including protein production and gene therapy.
The Cap structure is an essential component in the development of mRNA vaccines and therapeutics. Synthetic mRNA technology relies on Cap1 to enhance stability, translational efficiency and reduces the innate immune recognition of mRNA by toll-like receptors (TLRs) and other immune sensors, minimizing unwanted immune activation. This prevents unwanted inflammatory responses, thereby improving the stability and effectiveness of the therapeutic mRNA in human cells.
|
Product |
Cap AG (3'-OMe-7mG) |
LZCap AG(3'Acm) |
AG (no capping) |
|
mRNA yield (μg) |
164 |
173 |
200 |
MS analysis of the capping efficiency of LZCapped Luciferase mRNA.
How’s the mRNA stability towards the decapping enzyme
mRNAs with LZCap AG(3'Acm) and LZCap AG M6 (3'Acm) show higher resistance towards the decapping enzyme (NEB). It is noted that CapM6 (3'-OMe-6mA-7mG) is also resistant to decapping enzym, but the regular Cap AG (3'-OMe-7mG) does not show the resistance.
How’s the binding affinity of LZCap to eIF4E?
How about the protein expression of LZCap AG(3'Acm) capped mRNA?
The expression of LZCap AG(3'Acm) capped luciferase mRNA is significantly higher than that of the Cap1 analog (3'-OMe-7mG) capped mRNA in different cell lines* (3T3-L1，Hela，JAWs， HEK293T and Huh7)，The 130 repetition experiment showed about a 1.5 times higher expression of LZCap AG(3'Acm) capped luciferase mRNA than the (3'-OMe-7mG) capped mRNA on average. Similar result is observed in mice. Protein expression level may vary slightly with different mRNA sequence.
Do you have more animal data?
The expression efficiency of LZCap AG(3'Acm) or LZCap AG(3'FMom) capped
GLuc encoding mRNA shows a higher in vivo expression relative to CapAG (3'-OMe-7mG) capped mRNAs in Cynomolgus Monkey and Pig.
How about the innate immunogenicity of LZCap AG capped mRNAs ?
LZCapAG ( 3'Acm ) capped mRNAs show low innate immunogenicity. TLR8, TLR7, IL-1A and B play an important role in the immune response induced by uncapped RNA. The in vivo studies of LZCapped mRNA immunogenicity analysis showed that uncapped RNA caused significant changes in the transcription level of immune-related factors in mice. Both LZCap and 3'-OMe-7mG capped mRNAs display similar lower immune factor transcription level in mice after a single RNA-stimulated injection.
Did you do some safety test?
Yes, we did Cytotoxicity Test, Human Polymerase Inhibition Study and Ames Test.
- There are no or little cytotoxicity observed for the nucleoside monomer of 3'-Acm-7mG（CC50＞10000nM）in 293T、Huh7、MRC5、THP1 and U87MG cells.
- Human DNA polymerase ( α ,β , γ and Klenow) and mitochondrial RNA polymerase (hPOLRMT) activity inhibition studies show that 3'-Acm-7mG TP does not inhibit human DNA or RNA polymerase.
- The bacterial reverse mutation test (Ames) detects associated genetic alterations, as well as genotoxic carcinogens in most rodents and humans. Ames test showed 3'-Acm-7mG has no genotoxicity.
Has this product been patented?
Yes. The patent has been granted in the USA.
Ordering information
| Product Name | Specifications | Catalog No. |
| LZCap AG(3'Acm)（ 100 mM） | 100μL, 1mL | 10684ES |
| LZCap AU(3'Acm)（ 100 mM） | 100μL, 1mL | 10685ES |
| LZCap GG(3'Acm)（ 100 mM） | 100μL, 1mL | 10686ES |
| LZCap AG(3'Ma-Cy5)（ 25 mM） | 100μL, 1mL | 10688ES |
| LZCap AG(3'Ma-Cy7)（ 25 mM） | 100μL, 1mL | 10689ES |
| LZCap (AG(3'Ma-Cy3) (25 mM) | 100μL, 1mL | 10687ES |
| LZCap (AG(3'Ma-Biotin) (25 mM) | 100μL, 1mL | Inquiry |
| LZCap (AG(3'Ma-Peg5-FAM) (25 mM) | 100μL, 1mL | Inquiry |
| LZCap (AG(3'Ma-C6-MANT) (25 mM) | 100μL, 1mL | Inquiry |
| LZCap (AG(3'Acm) Firefly luc mRNA (1μg/μL) | 100μL, 1mL | Inquiry |
| LZCap (AG(3'Acm) eGFP mRNA (1μg/μL) | 100μL, 1mL | Inquiry |
| LZCap (AG(3'Acm) eGFP saRNA (1μg/μL) | 100μL, 1mL | Inquiry |
| LZCap (AG(3'Acm) RFP mRNA (1 μg/μL) | 100μL, 1mL | Inquiry |
| LZCap (AG(3'Acm) Cas9 mRNA (1μg/μL) | 100μL, 1mL | Inquiry |
| LZCap (AG(3'Acm) Gluc mRNA (1μg/μL) | 100μL, 1mL | Inquiry |
Reference:
- Molecular mechanisms of coronavirus RNA capping and methylation, Virologica Sinica 31(1)
- mRNA vaccines — a new era in vaccinology. Nat. Rev. Drug. Discov. 17, 261–279 (2018).
- Translation Initiation Regulated by RNA-Binding Protein in Mammals: TheModulation of Translation Initiation Complex by Trans-Acting Factors, Cells 2021, 10(7), 1711