Towards mRNA with superior translational activity: synthesis and properties of ARCA tetraphosphates with single phosphorothioate modifications

Abstract

We describe the chemical synthesis and preliminary biophysical and biochemical characterization of a series of mRNA 5' end (cap) analogs designed as reagents for obtaining mRNA molecules with augmented translation efficiency and stability in vivo and as useful tools to study mRNA metabolism. The analogs share three structural features: (i) 5',5'- bridge elongated to tetraphosphate to increase their affinity to translation initiation factor eIF4E (ii) a single phosphorothioate modification at either the α, β, γ or δ-position of the tetraphosphate to decrease their susceptibility to enzymatic degradation and/or to modulate their interaction with specific proteins and (iii) a 2'-O-methyl group in the ribose of 7-methylguanosine, characteristic to Anti-Reverse Cap Analogs (ARCAs), which are incorporated into mRNA during in vitro transcription exclusively in the correct orientation. The dinucleotides bearing modified tetraphosphate bridge were synthesized by ZnCl(2) mediated coupling between two mononucleotide subunits with isolated yields of 30-65%. The preliminary biochemical results show that mRNAs capped with new analogs are 2.5-4.5 more efficiently translated in a cell free system than m(7)GpppG-capped mRNAs, which makes them promising candidates for RNA-based therapeutic applications such as gene therapy and anti-cancer vaccines.

Fig. 1

Cap structures. A) A general depiction of the eukaryotic mRNA 5' end. B) Structures of standard triphosphate analogs: m⁷GpppG and m₂^7,3'-OGpppG (ARCA). C) Structures of m₂^7,2'-OGppppG and analogs synthesized in this study.

Fig. 2

Typical HPLC profiles of reaction mixtures. A) Progress in synthesis of 1 after 1 h and 4 h. B) Synthesis of 2 after 3 days.

Fig. 3

K_AS constants for eIF4E - cap analog complexes. A) Fluorescence quenching curves for titration of eIF4E with selected cap analogs (m₂^7,3'-OGpppG, m₂^7,2'-OGppppG and 4a). B) Comparison of K_AS values of 4P-S-ARCAs (1 – 4) with standard triphosphate analogs and unmodified tetraphosphate ARCA. The K_AS values are means of 3 experiments

Fig. 4

RP HPLC profiles of mRNA cap dinucleotides digestion by hDcpS. A) m⁷GpppG B, m₂^7,2'-OGppppG C) 1a D) 1b E) 4a F) 4b. Assay conditions are given in the Experimental Section

Fig. 5

Translational efficiency of mRNA transcripts in vitro. A) Example experiment of translational efficiency of mRNA transcript encoding firefly luciferase capped with various dinucleotides. B) Comparison of relative cap-dependent translational efficiency of mRNA transcripts. The mean values were calculated from 2–3 assay repetitions for each of two independent mRNA syntheses.

Scheme 1

The synthesis of tetraphosphate S-ARCA 1–4. A) Synthesis of 1 and 4 by the "3 + 1*" strategy. B) Synthesis of 2 and 3 by the "2 + 2*" strategy. C) Synthesis of 2 by the "3 + 1*" strategy (the asterisk denotes the activated nucleotide subunit). Reagents: i. 1) PSCl₃, trimethylphosphate, 2,6-lutidine, 0 °C 2) 0.5M tributylammonium pyrophosphate in DMF; ii. imidazole, 2,2'-dithiodipyridine, PPh₃, DMF; iii. ZnCl₂, DMF; iv. triethylammonium phosphate, ZnCl₂, DMF; v. triethylammonium thiophosphate, ZnCl₂, DMF

Scheme 2

Possible disconnections of tetraphosphate bridged thio-modified dinucleotides. A) Tetraphosphates with phosphorothioate modification in the external position. B) Tetraphosphates with phosphorothioate modification in the internal position