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Nucleotides for mRNA Modification

Synthetic messenger RNA (mRNA) can be used to deliver exogenous genetic information inside cells that is converted by the cellular translational machinery into the encoded protein which subsequently induces a cellular response depending on its biological function.

Therapeutic protein encoding mRNAs have most recently gained increasing attention as versatile protein delivery molecules in non-viral gene therapy mostly due to their superior safety profile compared to plasmid DNA-based approaches (inability to integrate into the host genome)[1]. In addition, high-purity mRNA is easily synthesized even large scales by phage RNA polymerase-mediated in vitro transcription[2,3].

Their application however, was so far been hampered by the limited stability and strong immunogenicity of in vitro transcribed mRNAs subsequently leading to increased expression rates of the respective protein.

Enzymatic incorporation of a set several nucleotide analogs (single or combined) during the transcription process markedly improves the pharmacokinetic properties of in vitro transcribed mRNA both in terms of stability (increased nuclease resistance) and decreased immunogenicity (Fig. 1)[4-8].

Find more Cap analogs in the 5'-Capping section!

in vitro TranscriptionARCAm5CTPm6ATPs2UTPΨme1Ψ5moUTP

Figure 1: Pharmacokinetic properties of synthetic mRNA are improved by nucleotide analog incorporation during in vitro RNA synthesis.
ARCA: "anti-reverse" cap analog, m5CTP = 5'-methyl-cytidine triphosphate, m6ATP=N6-methyl-adenosine-5'-triphosphate, s2UTP = 2-thio-uridine triphosphate, Ѱ = pseudouridine triphosphate, me1Ѱ = N1Methylpseudouridine triphosphate, 5moUTP = 5-Methoxyuridine triphosphate

For bulk inquiries please contact us at nucleotides@jenabioscience.com

Selected References

[1] Tavernier et al. (2011) mRNA as gene therapeutic: How to control protein expression. Journal of controlled disease 150:238.
[2] Karikó et al. (2011) Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA. Nuclei Acids Res. 39 (21):9329.
[3] Pascolo et al. (2006) Vaccination with messenger RNA. In: Methods in Molecular Medicine 127 (Saltzmann). Humana Press.
[4] Kormann et al. (2011) Expression of therapeutic proteins after delivery of chemically modified mRNA in mice. Nature Biotechnology 29 (2):154.
[5] Karikó et al. (2008) Incorporation of Pseudouridine into mRNA Yields Superior Nonimmunogenic Vector With Increased Translational Capacity and Biological Stability. Mol. Ther. 16 (11):1833.
[6] Karikó et al. (2005) Suppression of RNA Recognition by Toll-like Receptors: The Impact of Nucleoside Modification and the Evolutionary Origin of RNA. Immunity 23:165.
[7] Anderson et al. (2011) Nucleoside modifications in RNA limit activation of 2'-5'-oligoadenylate synthetase and increase resistance to cleavage by RNAse L. Nuclei Acids Res. 39 (21):9329.
[8] Warren et al. (2011) Highly Efficient Reprogramming to Pluripotency and Directed Differentiation of Human Cells with Synthetic Modified mRNA. Cell Stem Cell 7:618.
[9] Andies et al. (2015) N (1)-methylpseudouridine-incorporated mRNA outperforms pseudouridine-incorporated mRNA by providing enhanced protein expression and reduced immunogenicity in mammalian cell lines and mice. J. Control. Release 217:337.
[10] Li et al. (2016) Effects of Chemically Modified Messenger RNA on Protein Expression Bioconjugate Chem. 27 (3):849.