The tRNA-Modification Geranyl-2-Thiouridine Enables RNA-Lipid Association

The packaging of RNA in lipid nanoparticles for protection and delivery was key for the development of mRNA vaccines and therapeutics. Formulation requires the addition of a variety of lipids (including cationic) in high amounts to form complexes with the negatively charged RNA. While this approach was successful for the development of RNA vaccines, it represents a major challenge for future developments. Increasing the lipophilicity of RNA is one possible approach to reducing the amount of lipid required. In nature, geranyl-2-thiouridine occurs at the "Wobble" position 34 of certain bacterial tRNAs. This work details investigations into geranylated oligonucleotides and their interactions with lipids. We developed an efficient post-synthetic procedure for oligonucleotide geranylation in aqueous solutions using a geranyl-sulfonium salt. We discovered that the geranyl-modification can be replaced by thiol-nucleophiles, enabling a reversible switch of the hydrophobic character of the oligonucleotides. We show that the geranyl-modified oligonucleotides anchor onto neutral membrane structures and oil droplets. Oligonucleotides bound to the outer surface of neutral liposomes can flip to the inner membrane surface by freeze-thaw cycles, leading to a protective effect against enzymatic or chemical degradation. Our work expands the scope of oligonucleotide lipidation strategies, which are of great interest in RNA delivery systems.