Abstract
mRNA-based therapies and vaccines constitute a disruptive technology with the potential to revolutionize modern medicine. Chemically modified 5′ cap structures have provided access to mRNAs with superior translational properties that could benefit the currently flourishing mRNA field. Prime examples of compounds that enhance mRNA properties are antireverse cap analog diastereomers that contain an O-to-S substitution within the β-phosphate (β-S-ARCA D1 and D2), where D1 is used in clinically investigated mRNA vaccines. The compounds were previously found to have high affinity for eukaryotic translation initiation factor 4E (eIF4E) and augment translation in vitro and in vivo. However, the molecular basis for the beneficial “thio-effect” remains unclear. Here, we employed multiple biophysical techniques and captured 11 cap analog-eIF4E crystallographic structures to investigate the consequences of the β-O-to-S or -Se substitution on the interaction with eIF4E. We determined the SP/RP configurations of β-S-ARCA and related compounds and obtained structural insights into the binding. Unexpectedly, in both stereoisomers, the β-S/Se atom occupies the same binding cavity between Lys162 and Arg157, indicating that the key driving force for complex stabilization is the interaction of negatively charged S/Se with positively charged amino acids. This was observed for all structural variants of the cap and required significantly different conformations of the triphosphate for each diastereomer. This finding explains why both β-S-ARCA diastereomers have higher affinity for eIF4E than unmodified caps. Binding affinities determined for di-, tri-, and oligonucleotide cap analogs suggested that the “thio-effect” was preserved in longer RNAs. Our observations broaden the understanding of thiophosphate biochemistry and enable the rational design of translationally active mRNAs and eIF4E-targeting drugs.
Highlights
Phosphorothioate (PS) modification ofnucleotides is one of the most frequently used synthetic approaches for investigating nucleic acid metabolism or for improving the pharmacokinetics of therapeutic or diagnostic molecules.[1−6] Many applications of thio-modified oligonucleotides, such as the elucidation of ion-binding sites on RNA7 and identification of phosphate sites important for ligand or protein binding or for catalysis,[8−12] assume that this modification has little or no effect on the secondary structure of nucleic acids
A series of therapeutically relevant βphosphorothioate-modified analogs of mRNA 5′ ending in complex with eukaryotic translation initiation factor 4E was structurally characterized
The local environment of sulfur or selenium formed by positively charged amino acids clearly suggests that the negative charge in the modified phosphate residues is mainly localized at the S/Se atoms
Summary
ABSTRACT: mRNA-based therapies and vaccines constitute a disruptive technology with the potential to revolutionize modern medicine. We determined the SP/RP configurations of β-S-ARCA and related compounds and obtained structural insights into the binding In both stereoisomers, the β-S/Se atom occupies the same binding cavity between Lys[162] and Arg[157], indicating that the key driving force for complex stabilization is the interaction of negatively charged S/Se with positively charged amino acids. This was observed for all structural variants of the cap and required significantly different conformations of the triphosphate for each diastereomer. Our observations broaden the understanding of thiophosphate biochemistry and enable the rational design of translationally active mRNAs and eIF4E-targeting drugs
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