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. 2016 Nov 16;44(20):9578-9590.
doi: 10.1093/nar/gkw896. Epub 2016 Oct 7.

Cap analogs modified with 1,2-dithiodiphosphate moiety protect mRNA from decapping and enhance its translational potential

Malwina Strenkowska  1 , Renata Grzela  2 , Maciej Majewski  1 , Katarzyna Wnek  1 , Joanna Kowalska  1 , Maciej Lukaszewicz  1 , Joanna Zuberek  1 , Edward Darzynkiewicz  1   2 , Andreas N Kuhn  3   4 , Ugur Sahin  3   4 , Jacek Jemielity  5
Affiliations

Cap analogs modified with 1,2-dithiodiphosphate moiety protect mRNA from decapping and enhance its translational potential

Malwina Strenkowska et al. Nucleic Acids Res. .

Abstract

Along with a growing interest in mRNA-based gene therapies, efforts are increasingly focused on reaching the full translational potential of mRNA, as a major obstacle for in vivo applications is sufficient expression of exogenously delivered mRNA. One method to overcome this limitation is chemically modifying the 7-methylguanosine cap at the 5' end of mRNA (m7Gppp-RNA). We report a novel class of cap analogs designed as reagents for mRNA modification. The analogs carry a 1,2-dithiodiphosphate moiety at various positions along a tri- or tetraphosphate bridge, and thus are termed 2S analogs. These 2S analogs have high affinities for translation initiation factor 4E, and some exhibit remarkable resistance against the SpDcp1/2 decapping complex when introduced into RNA. mRNAs capped with 2S analogs combining these two features exhibit high translation efficiency in cultured human immature dendritic cells. These properties demonstrate that 2S analogs are potentially beneficial for mRNA-based therapies such as anti-cancer immunization.

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Figures

Figure 1.
Figure 1.
Synthesis of dithiodiphosphate cap analogs. (A) Structures of dithiodiphosphate cap precursors synthesized in this study. (B) Scheme of dithiodiphosphate cap synthesis. Reagents included (i) MgCl2, microwave radiation (MW), dimethylformamide (DMF); and (ii) 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU), dithiothreitol (DTT), MW, DMF. R = H, CH3. Combinations of substrates used in final coupling included combination 1: m = 1, n = 0; combination 2: m = 1, n = 1; and combination 3: m = 2, n = 0.
Figure 2.
Figure 2.
Dithiodiphosphate cap analogs and their selected characteristic properties. (A) Structures of dithiodiphosphate cap analogs synthesized in this study. (B) During synthesis, two new P-chiral centers (indicated by *) are generated, leading to four diastereoisomeric forms of each cap analog (D1–D4). Diastereoisomers were separated by semi-preparative RP-HPLC, resulting in the following final compounds/mixtures: m7GppSpSG (3): D1 (3a), D2 (3b), D3 (3c), D4 (3d); m27,2′-OGppSpSG (4): D1D2 (4a), D3 (4b), D4 (4c); m27,2′-OGppSpSpG (5): D1D2 (5a), D3D4 (5b); m7GpppSpSG (6): D1D2 (6a), D3D4 (6b); m27,2′-OGpppSpSG (7): D1D2 (7a), D3D4 (7b). (C) An example 31P NMR spectrum of compound 7a displays the characteristic shifts of P-signals as the result of sulfur substitutions. In the spectrum, signals of P atoms of two diastereoisomers (D1+D2) are present and partially overlap. Dual signals from diastereoisomeric forms are marked here with a preceding ‘2’; for example, ‘2d’ means that two doublet signals of the same P nucleus from both diastereoisomers overlap.
Figure 3.
Figure 3.
Expression of analog-capped mRNAs in human immature dendritic cells (hiDCs). Luciferase activity was measured for 72 h following electroporation of mRNA containing the respective tetraphosphate (A) or triphosphate (B) 5′-cap into hiDCs. In each experiment, m27,2′-OGppSpG (D1) was used as a reference. Duplicate measurements were performed and the average bioluminescence signal (±s.d.) is shown as a function of time.
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References

    1. Quabius E., Krupp G. Synthetic mRNAs for manipulating cellular phenotypes: an overview. N. Biotechnol. 2014;32:229–235. - PubMed
    1. Deering R., Kommareddy S. Nucleic acid vaccines: prospects for non-viral delivery of mRNA vaccines. Expert Opin. Drug Deliv. 2014;11:885–899. - PubMed
    1. Saunders A., Wang J. Export and expression: mRNAs deliver new messages for controlling pluripotency. Cell Stem Cell. 2014;14:549–550. - PubMed
    1. Sahin U., Karikó K., Türeci Ö. mRNA-based therapeutics—developing a new class of drugs. Nat. Rev. Drug Discov. 2014;13:759–780. - PubMed
    1. Astakhova I.K., Wengel J. Scaffolding along nucleic acid duplexes using 2′-amino-locked nucleic acids. Acc. Chem. Res. 2014;47:1768–1777. - PubMed