Trinucleotide mRNA Cap Analogue N 6-Benzylated at the Site of Posttranscriptional m6 A m Mark Facilitates mRNA Purification and Confers Superior Translational Properties In Vitro and In Vivo.

Autor: Warminski M; Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, 02-089 Warsaw, Poland., Trepkowska E; Explorna Therapeutics sp. z o.o. Zwirki i Wigury 93, 02-089 Warsaw, Poland., Smietanski M; Explorna Therapeutics sp. z o.o. Zwirki i Wigury 93, 02-089 Warsaw, Poland., Sikorski PJ; Centre of New Technologies, University of Warsaw, 02-089 Warsaw, Poland.; Laboratory of Epitranscriptomics, Department of Environmental Microbiology and Biotechnology, Institute of Microbiology, Faculty of Biology, Biological and Chemical Research Centre, University of Warsaw, 02-089 Warsaw, Poland., Baranowski MR; Explorna Therapeutics sp. z o.o. Zwirki i Wigury 93, 02-089 Warsaw, Poland., Bednarczyk M; Centre of New Technologies, University of Warsaw, 02-089 Warsaw, Poland.; Explorna Therapeutics sp. z o.o. Zwirki i Wigury 93, 02-089 Warsaw, Poland., Kedzierska H; Explorna Therapeutics sp. z o.o. Zwirki i Wigury 93, 02-089 Warsaw, Poland., Majewski B; Explorna Therapeutics sp. z o.o. Zwirki i Wigury 93, 02-089 Warsaw, Poland., Mamot A; Centre of New Technologies, University of Warsaw, 02-089 Warsaw, Poland., Papiernik D; Explorna Therapeutics sp. z o.o. Zwirki i Wigury 93, 02-089 Warsaw, Poland., Popielec A; Explorna Therapeutics sp. z o.o. Zwirki i Wigury 93, 02-089 Warsaw, Poland., Serwa RA; Proteomics Core Facility, IMol Polish Academy of Sciences, 02-247 Warsaw, Poland., Shimanski BA; Department of Chemistry & Biochemistry, University of Delaware, Newark, Delaware 19716, United States., Sklepkiewicz P; Explorna Therapeutics sp. z o.o. Zwirki i Wigury 93, 02-089 Warsaw, Poland., Sklucka M; Explorna Therapeutics sp. z o.o. Zwirki i Wigury 93, 02-089 Warsaw, Poland., Sokolowska O; Explorna Therapeutics sp. z o.o. Zwirki i Wigury 93, 02-089 Warsaw, Poland., Spiewla T; Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, 02-089 Warsaw, Poland.; Explorna Therapeutics sp. z o.o. Zwirki i Wigury 93, 02-089 Warsaw, Poland., Toczydlowska-Socha D; Explorna Therapeutics sp. z o.o. Zwirki i Wigury 93, 02-089 Warsaw, Poland., Warminska Z; Centre of New Technologies, University of Warsaw, 02-089 Warsaw, Poland., Wolosewicz K; Explorna Therapeutics sp. z o.o. Zwirki i Wigury 93, 02-089 Warsaw, Poland., Zuberek J; Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, 02-089 Warsaw, Poland., Mugridge JS; Department of Chemistry & Biochemistry, University of Delaware, Newark, Delaware 19716, United States., Nowis D; Explorna Therapeutics sp. z o.o. Zwirki i Wigury 93, 02-089 Warsaw, Poland.; Laboratory of Experimental Medicine, Faculty of Medicine, Medical University of Warsaw, 02-097 Warsaw, Poland., Golab J; Explorna Therapeutics sp. z o.o. Zwirki i Wigury 93, 02-089 Warsaw, Poland.; Laboratory of Experimental Medicine, Faculty of Medicine, Medical University of Warsaw, 02-097 Warsaw, Poland., Jemielity J; Centre of New Technologies, University of Warsaw, 02-089 Warsaw, Poland.; Explorna Therapeutics sp. z o.o. Zwirki i Wigury 93, 02-089 Warsaw, Poland., Kowalska J; Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, 02-089 Warsaw, Poland.; Explorna Therapeutics sp. z o.o. Zwirki i Wigury 93, 02-089 Warsaw, Poland.
Jazyk: angličtina
Zdroj: Journal of the American Chemical Society [J Am Chem Soc] 2024 Mar 27; Vol. 146 (12), pp. 8149-8163. Date of Electronic Publication: 2024 Mar 05.
DOI: 10.1021/jacs.3c12629
Abstrakt: Eukaryotic mRNAs undergo cotranscriptional 5'-end modification with a 7-methylguanosine cap. In higher eukaryotes, the cap carries additional methylations, such as m6 A m ─a common epitranscriptomic mark unique to the mRNA 5'-end. This modification is regulated by the Pcif1 methyltransferase and the FTO demethylase, but its biological function is still unknown. Here, we designed and synthesized a trinucleotide FTO-resistant N 6-benzyl analogue of the m6 A m -cap-m 7 Gppp Bn6 A m pG (termed AvantCap ) and incorporated it into mRNA using T7 polymerase. mRNAs carrying Bn6 A m showed several advantages over typical capped transcripts. The Bn6 A m moiety was shown to act as a reversed-phase high-performance liquid chromatography (RP-HPLC) purification handle, allowing the separation of capped and uncapped RNA species, and to produce transcripts with lower dsRNA content than reference caps. In some cultured cells, Bn6 A m mRNAs provided higher protein yields than mRNAs carrying A m or m6 A m , although the effect was cell-line-dependent. m 7 Gppp Bn6 A m pG-capped mRNAs encoding reporter proteins administered intravenously to mice provided up to 6-fold higher protein outputs than reference mRNAs, while mRNAs encoding tumor antigens showed superior activity in therapeutic settings as anticancer vaccines. The biochemical characterization suggests several phenomena potentially underlying the biological properties of AvantCap : (i) reduced propensity for unspecific interactions, (ii) involvement in alternative translation initiation, and (iii) subtle differences in mRNA impurity profiles or a combination of these effects. AvantCapped- mRNAs bearing the Bn6 A m may pave the way for more potent mRNA-based vaccines and therapeutics and serve as molecular tools to unravel the role of m6 A m in mRNA.
Databáze: MEDLINE
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