Messenger RNA (mRNA) was first discovered in the 1960s, but its instability and immunogenicity have severely hindered the development of mRNA therapy. To solve this problem, researchers have contrived several mRNA modification strategies, among which nucleoside modification plays an important role. It can effectively reduce the immunogenicity of mRNA and inhibit innate immune activation, making mRNA a powerful tool for regenerative medicine and cell reprogramming.
1.Robust evidence reveals that mRNA therapy has unparalleled advantages:
1.1 mRNA does not need to enter the nucleus to function
Once entering the cytoplasm, the mRNA initiates protein translation instantly. On the contrary, DNA needs to enter the nucleus to complete transcription and the generated mRNA can only play its role after exiting the nucleus, which results in lower efficiency.
1.2 Higher security
Compared to viral vectors, mRNA does not integrate into the genome but only expresses the encoded protein instantaneously, so it has higher safety.
1.3 mRNA can be synthesized directly by in vitro transcription (IVT) process.
The process is relatively simple and inexpensive, allowing rapid response to multiple outbreaks.
The autoimmunogenicity of mRNA transcribed in vitro is a rather serious issue since exogenous RNA can be regarded as the signal of viral infection. Nucleoside chemical modification strategies can reduce immunogenicity without affecting its translational properties. For instance, natural adenosine is replaced with N1-methyladenosine (m1A) or N6-methyladenosine (m6A); natural cytidine is replaced with 5-methylcytidine (m5C); natural uridine is replaced by 5-methoxyuridine (5moU), N1-methyl-pseudouridine (m1Ψ), and pseudouridine (Ψ). Among them, N1-methyl-pseudouridine (m1Ψ), 5-methoxyuridine (5moU), and pseudouridine (Ψ) have attracted much attention, because both in vivo and in vitro experiments show that they can effectively reduce the immunogenicity of mRNA and improve the translation efficiency.
2.Introduction to modified nucleosides
Pseudouridine, the first modified nucleoside discovered 70 years ago, is derived from uridine through base isomeric reaction, in which the nuclear base rotates 180° around the N3-C6 axis, resulting in a change in the nuclear base-sugar bond (from N1-C1′ to C5-C1′). The resulting C-C bond allows the nuclear bases to rotate more freely. Pseudouridine can base pair with adenosine like uridine, but it can alter RNA structure by improving base pairing, base stacking, and backbone stability. In 2005, Katalin Kariko et al. found that the introduction of pseudouridine into RNA can reduce its immunogenicity, and the immunogenicity of RNA decreases with the increase of the introduction proportion of pseudouridine. In 2008, Katalin Kariko et al. also found that completely replacing uridine with pseudouridine can improve mRNA stability and enhance its translation ability.
2.2 N1-Methyl-Pseudouridine (m1Ψ)
N1-methyl-pseudouridine is a methylpseudouridine, an N1-modified pseudouridine derivative, which is a natural modification found in 18S rRNA and tRNA in many organisms. N1-methyl-pseudouridine has a methyl group at the N1 position, thus eliminating the additional hydrogen bond donor. Pseudouridine and N1-methyl-pseudouridine share a key common feature, the C5-C1′ bond, which enables rotation between the nuclear base and the sugar portion, and may help improve base pairing, base stacking, and double-stranded stability. N1-methyl-pseudouridine pairs with A with a higher affinity than uridine and is less likely to activate PKR, thus having better translation efficiency. N1-methyl-pseudouridine, which is structurally similar to pseudouridine, may also enable mRNA to evade immune responses. In 2015, Oliwia Andries et al. found that the complete replacement of uridine with N1-methyl-pseudouridine could reduce mRNA immunogenicity and enhance mRNA protein expression ability compared to the complete replacement of uridine with pseudouridine.
As early as the 1970s, scientists identified m6A modifications in RNA, the most abundant internal modifications of mRNAs and long non-coding RNAs in most eukaryotes. In 2012, scientists showed that m6A modification was related to mRNA stability, splicing processing, and translation. In 2018, Shinichiro Akichikade et al. also found that m6A could promote mRNA translation.
5-methylcytosine (m5C) has been found in mRNA, rRNA, and tRNA of a variety of representative organisms. As a reversible epigenetic modification, C modification of m5RNA can affect the fate of the modified RNA molecules, including promoting mRNA stability, splicing, and nuclear transport. Viral protein expression, DNA damage repair, mRNA stability, cell tolerance, proliferation and migration, stem cell development, differentiation, and reprogramming are also affected. In addition, the distribution of m5C varies by cell type. Therefore, its modifications at specific sites of mRNAs show different regulatory activities.
5-methoxyuridine (5moU) is a rare nucleoside. Adding 5-methoxyuridine to RNA (mRNA) can reduce the immunogenicity of the resulting mRNA. In 2022, Hanieh Moradian et al. found that chemical modifications of uridine, specifically 5-methoxyuridine, showed the highest levels of protein production while inducing a negligible response to inflammatory macrophages.
3. Significant applications of modified nucleosides – in COVID-19 mRNA vaccines
Pfizer-BioNTech and Moderna Therapeutics developed two new mRNA-based vaccine platforms (comirnaty® and spikevax®) in 2020 and were the first to achieve 90%+ efficacy. Both were composed of N1-methyl-pseudouridine modified mRNA encoding SARS-COVID-19 spike protein and were delivered using lipid nanoparticle (LNP) preparations.
The success of the LNP was quickly hailed by many as the unsung hero of the COVID-19 mRNA vaccine since the problems in the delivery of ribonucleic acid have plagued people for decades. However, research results from clinical trials of the Curevac mRNA vaccine (CVnCoV) suggest that the delivery system is not the only reason for success. CVnCoV consists of unmodified mRNA (encoding the same spike protein as Moderna’s and Pfizer-BioNTech’s mRNA vaccines) and is formulated with the same LNP as Pfizer-BioNTech’s vaccine (Acuitas ALC-0315). Yet, its efficacy is only 48%. The significant difference in efficacy could be attributed to the presence of a key RNA modification (N1-methyl-pseudouridine) in Pfizer-BioNTech’s and Moderna’s mRNA vaccines.
N1-methyl-pseudouridine, instead of conventional UTP, can reduce the immunogenicity of the generated mRNA and increase its expression. Human cells contain a variety of pattern recognition receptors, which can recognize pathogenic RNAs and activate downstream signaling pathways in response. For example, intracellular receptors TLR3, TLR7, and TLR8 recognize double- and single-stranded RNAs. Cytoplasmic receptors RIG-I and MDA-5 recognize double-stranded and 5′-triphosphate modified RNAs. The purpose of an mRNA vaccine is to produce an antigenic protein and then activate the corresponding immune response. If the mRNA induces a strong immune response before it is translated into an antigenic protein, it may inhibit the expression of the antigen and thus reduce the effectiveness of the vaccine, or even cause allergic reactions to harm the human body.
Overall, numerous studies have shown that RNAs produced using specially modified nucleosides are safer and more effective. The mRNAs produced by N1-methyl-pseudouridine can greatly improve the effectiveness in both aspects of reducing immunogenicity and improving translation efficiency.
Related Products:
| Name | CAS | Description |
| pseudoUridine and Its Derivatives | BOC Sciences has become the leading supplier of PseudoUridine with the most capacity in the world. | |
| 1-Methyladenosine | 15763-06-1 | 1-methyladenosine is a methyladenosine carrying a methyl substituent at position 1. |
| N6-Methyladenine | 443-72-1 | N6-methyladenosine (m6A) is methylation that occurs in the N6-position of adenosine. |
| 5-methylcytidine | 2140-61-6 | 5-Methylcytidine is a derivative of Cytidine, found in ribonucleic acids of animals, plants and bacteria. |
| 5-methoxyuridine | 35542-01-9 | 5-Methoxyuridine is an analog of Uridine and is used as a reagent in the synthesis of 5-OMe-UDP, a potent and selective P2Y6-receptor agonist. |
| N1-MethylpseudoUridine | 13860-38-3 | N1-methyl-pseudoUridine (1-Methylpseudouridine), a methylpseudoUridine, outperforms 5 mC and 5 mC/N1-methyl-pseudoUridine in translation. |
| β-pseudoUridine | 1445-07-4 | An isomer of the nucleoside uridine found in all species and in many classes of RNA except mRNA. |