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 AntiReverse 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.

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

“…The translation efficiency of differently capped luciferase transcripts was determined in Flexi RRL (Promega) as described previously (24). Briefly, the translation reactions were performed in 10 μl volume for 60 min at 30°C under conditions determined for cap-dependent translation.…”

“…Therefore, we previously conducted a systematic search for cap analogs modified within the oligophosphate bridge to identify those with properties similar or superior to β-S-ARCA. Among these analogs is a group with a single O-to-S substitution within a tetraphosphate bridge (4P-S-ARCAs) (24). Due to the presence of an additional phosphate group, these analogs exhibit a higher affinity to eIF4E than 3P-S-ARCAs and RNAs capped with these 4P analogs are efficiently translated in rabbit reticulocyte lysates (RRLs).…”

“…Due to the presence of an additional phosphate group, these analogs exhibit a higher affinity to eIF4E than 3P-S-ARCAs and RNAs capped with these 4P analogs are efficiently translated in rabbit reticulocyte lysates (RRLs). However, preliminary studies indicated that the single O-to-S substitution in the tetraphosphate bridge (either β or γ position) provides only partial protection against Dcp2 (24), which could limit in vivo applications.…”

Phosphate-modified analogues of m 7 GTP and m 7 Gppppm 7 G—Synthesis and biochemical properties

“…[37][38][39][40] A correlation between the RP-HPLC elution order, H8 proton shift and the absolute configuration has been reported for various purine NTPaS, NDPaS, NTPaBH 3 , NDPaBH 3 41 and related compounds. 42 For example, the faster eluting diastereomers of ATPaS and ATPaBH 3 have less shielded H8 protons compared to corresponding slower eluting isomers, and the same stereochemistry (i.e., S P and R P configurations at a-phosphorus, respectively, see Fig. 2).…”

“…Translation efficiency experiments were performed as described previously. 42 Shortly, capped and polyadenylated luciferase mRNAs were synthesised in vitro on a PCR-amplified dsDNA template containing: SP6 promoter sequence, 5 0 UTR sequence of rabbit b-globin, luciferase ORF and a string of 31 adenosines. A typical in vitro transcription reaction mixture (40 ml) contained: SP6 transcription buffer (Fermentas), 0.7 mg of DNA template, 1 U/ml RiboLock Ribonuclease Inhibitor (Fermentas), 0.5 mM of each NTP and 0.1 mM GTP and 0.5 mM dinucleotide cap analogue (molar ratio cap analogue/GTP 5:1).…”