Recent COVID-19 vaccines unleashed the potential of mRNA-based therapeutics. A common bottleneck across mRNA-based therapeutic approaches is the rapid design of mRNA sequences that are translationally efficient, long-lived and non-immunogenic. Currently, an accessible software tool to aid in the design of such high-quality mRNA is lacking. Here, we present mRNAid, an open-source platform for therapeutic mRNA optimization, design and visualization that offers a variety of optimization strategies for sequence and structural features, allowing one to customize desired properties into their mRNA sequence. We experimentally demonstrate that transcripts optimized by mRNAid have characteristics comparable with commercially available sequences. To encompass additional aspects of mRNA design, we experimentally show that incorporation of certain uridine analogs and untranslated regions can further enhance stability, boost protein output and mitigate undesired immunogenicity effects. Finally, this study provides a roadmap for rational design of therapeutic mRNA transcripts.

Bioinformatics MSD Singapore 138665 Singapore

Department of Informatics and Chemistry Faculty of Chemical Technology University of Chemistry and Technology Prague 160 00 Czech Republic

Discovery Informatics MSD Czech Republic s r o Prague 150 00 Czech Republic

Quantitative Biosciences MSD Singapore 138665 Singapore

See more in PubMed

Pardi N., Hogan M.J., Porter F.W., Weissman D. mRNA vaccines—a new era in vaccinology. Nat. Rev. Drug Discovery. 2018; 17:261–279. PubMed PMC

Pastor F., Berraondo P., Etxeberria I., Frederick J., Sahin U., Gilboa E., Melero I.. An RNA toolbox for cancer immunotherapy. Nat. Rev. Drug Discovery. 2018; 17:751–767. PubMed

Lim S., Khoo R., Juang Y.-C., Gopal P., Zhang H., Yeo C., Peh K.M., Teo J., Ng S., Henry B.et al. .. Exquisitely specific anti-KRAS biodegraders inform on the cellular prevalence of nucleotide-loaded states. ACS Cent. Sci. 2020; 7:274–291. PubMed PMC

Corbett K.S., Edwards D.K., Leist S.R., Abiona O.M., Boyoglu-Barnum S., Gillespie R.A., Himansu S., Schäfer A., Ziwawo C.T., DiPiazza A.T.et al. .. SARS-CoV-2 mRNA vaccine design enabled by prototype pathogen preparedness. Nature. 2020; 586:567–571. PubMed PMC

Martini G.V.P., Guey L.T.. A new era for rare genetic diseases: messenger RNA therapy. Hum. Gene Ther. 2019; 30:1180–1189. PubMed

Hewitt S.L., Bai A., Bailey D., Ichikawa K., Zielinski J., Karp R., Apte A., Arnold K., Zacharek S.J., Iliou M.S.et al. .. Durable anticancer immunity from intratumoral administration of IL-23, IL-36γ, and OX40L mRNAs. Sci. Transl. Med. 2019; 11:eaat9143. PubMed

Damase T.R., Sukhovershin R., Boada C., Taraballi F., Pettigrew R.I., Cooke J.P.. The limitless future of RNA therapeutics. Front. Bioeng. Biotechnol. 2021; 9:628137. PubMed PMC

Jain R., Frederick J.P., Huang E.Y., Burke K.E., Mauger D.M., Andrianova E.A., Farlow S.J., Siddiqui S., Pimentel J., Cheung-Ong K.et al. .. MicroRNAs enable mRNA therapeutics to selectively program cancer cells to self-destruct. Nucleic Acid Ther. 2018; 28:285–296. PubMed PMC

Verma M., Choi J., Cottrell K.A., Lavagnino Z., Thomas E.N., Pavlovic-Djuranovic S., Szczesny P., Piston D.W., Zaher H.S., Puglisi J.D.et al. .. A short translational ramp determines the efficiency of protein synthesis. Nat. Commun. 2019; 10:5774. PubMed PMC

Vaidyanathan S., Azizian K.T., Haque A.K.M.A., Henderson J.M., Hendel A., Shore S., Antony J.S., Hogrefe R.I., Kormann M.S.D., Porteus M.H.et al. .. Uridine depletion and chemical modification increase Cas9 mRNA activity and reduce immunogenicity without HPLC purification. Mol. Ther. Nucleic Acids. 2018; 12:530–542. PubMed PMC

Gould N., Hendy O., Papamichail D. Computational tools and algorithms for designing customized synthetic genes. Front. Bioeng. Biotechnol. 2014; 2:41. PubMed PMC

Zhang H., Zhang L., Lin A., Xu C., Li Z., Liu K., Liu B., Ma X., Zhao F., Yao W.et al. .. Algorithm for optimized mRNA design improves stability and immunogenicity. Nature. 2023; 621:396–403. PubMed PMC

Lee J., Kladwang W., Lee M., Cantu D., Azizyan M., Kim H., Limpaecher A., Gaikwad S., Yoon S., Treuille A.et al. .. RNA design rules from massive open laboratory. Proc. Natl Acad. Sci. USA. 2014; 111:2122–2127. PubMed PMC

Kariko K., Muramatsu H., Welsh F.A., Ludwig J., Kato H., Akira S., Weissman D.. Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability. Mol. Ther. 2008; 16:1833–1840. PubMed PMC

Kormann M.S.D., Hasenpusch G., Aneja M.K., Nica G., Flemmer A.W., Herber-Jonat S., Huppmann M., Mays L.E., Illenyi M., Schams A.et al. .. Expression of therapeutic proteins after delivery of chemically modified mRNA in mice. Nat. Biotechnol. 2011; 29:154–159. PubMed

Zulkower V., Rosser S.. DNA Chisel, a versatile sequence optimizer. Bioinformatics. 2020; 36:4508–4509. PubMed

Alexaki A., Kames J., Holcomb D.D., Athey J., Santana-Quintero L.v., Lam P.V.N., Hamasaki-Katagiri N., Osipova E., Simonyan V., Bar H.et al. .. Codon and codon-pair usage tables (CoCoPUTs): facilitating genetic variation analyses and recombinant gene design. J. Mol. Biol. 2019; 431:2434–2441. PubMed

Sharp P.M., Li W.H.. The codon adaptation index—a measure of directional synonymous codon usage bias, and its potential applications. Nucleic Acids Res. 1987; 15:1281–1295. PubMed PMC

Hofacker I.L. Vienna RNA secondary structure server. Nucleic Acids Res. 2003; 31:3429–3431. PubMed PMC

Gaspar P., Moura G., Santos M.A.S., Oliveira J.L.. mRNA secondary structure optimization using a correlated stem–loop prediction. Nucleic Acids Res. 2013; 41:5490. PubMed PMC

Jeong D.-E., McCoy M., Artiles K., Ilbay O., Fire A., Nadeau K., Park H., Betts B., Boyd S., Hoh R.et al. .. Assemblies-of-Putative-SARS-CoV2-Spike-Encoding-mRNA-Sequences-for-Vaccines-BNT-162b2-and-mRNA-1273. (1 March 2022, date last accessed)https://virological.org/t/assemblies-of-putative-sars-cov2-spike-encoding-mrna-sequences-for-vaccines-bnt-162b2-and-mrna-1273/663.

World Health Organization Messenger RNA Encoding the Full-length SARS-CoV-2 Spike Glycoprotein. (1 March 2022, date last accessed)https://web.archive.org/web/20210105162941/https://mednet-communities.net/inn/db/media/docs/11889.doc.

Hale R.S., Thompson G.. Codon optimization of the gene encoding a domain from human type 1 neurofibromin protein results in a threefold improvement in expression level in Escherichia coli. Protein Expr. Purif. 1988; 12:185–188. PubMed

Diambra L.A. Differential bicodon usage in lowly and highly abundant proteins. PeerJ. 2017; 5:e3081. PubMed PMC

Gutman G.A., Hatfield G.W.. Nonrandom utilization of codon pairs in Escherichia coli. Proc. Natl Acad. Sci. USA. 1989; 86:3699–3703. PubMed PMC

Tuller T., Waldman Y.Y., Kupiec M., Ruppin E.. Translation efficiency is determined by both codon bias and folding energy. Proc. Natl Acad. Sci. USA. 2010; 107:3645–3650. PubMed PMC

Mauger D.M., Cabral J., Presnyak V., Su S.V., Reid D.W., Goodman B., Link K., Khatwani N., Reynders J., Moore M.J.et al. .. mRNA structure regulates protein expression through changes in functional half-life. Proc. Natl Acad. Sci. USA. 2019; 116:24075–24083. PubMed PMC

Leppek K., Byeon G.W., Kladwang W., Wayment-Steele H.K., Kerr C.H., Xu A.F., Kim D.S., Topkar V.V., Choe C., Rothschild D.et al. .. Combinatorial optimization of mRNA structure, stability, and translation for RNA-based therapeutics. Nat. Commun. 2022; 13:1536–1558. PubMed PMC

Mortimer S.A., Kidwell M.A., Doudna J.A.. Insights into RNA structure and function from genome-wide studies. Nat. Rev. Genet. 2014; 15:469–479. PubMed

Chung B.K.S., Lee D.Y.. Computational codon optimization of synthetic gene for protein expression. BMC Syst. Biol. 2012; 6:134–148. PubMed PMC

Ramanathan A., Robb G.B., Chan S.H.. mRNA capping: biological functions and applications. Nucleic Acids Res. 2016; 44:7511–7526. PubMed PMC