Towards the enzymatic labelling of oligonucleotides with triantennary N-acetyl galactosamine

The inclusion of chemical modifications into oligonucleotides stabilizes their backbones against nuclease mediated degradation and improves their biological activity. Even though cellular, tissue, and organ specific delivery remains a challenging undertaking, conjugation to triantennary N-acetyl galactosamine (GalNAc) ligands permits efficient delivery of nucleic acids to hepatocytes. GalNAc ligands are usually added to the termini of therapeutic oligonucleotides by costly and time-consuming chemical methods that often require specialized equipment and knowledge. Here, we have explored the possibility of enzymatically labelling oligonucleotides with triantennary GalNAc ligands which would be directly amenable to any type of sequence without requiring any synthetic effort. We have modified the gamma-phosphate of ATP with a GalNAc moiety and explored the possibility of transferring the ligand to 5’-termini of oligonucleotides using kinases. In parallel, we have equipped the 3’-position of an LNA nucleoside triphosphate with a GalNAc residue and used this nucleotide analog for labelling of oligonucleotides with template-independent polymerases. While kinases do not seem to tolerate the presence of such a bulky residue on ATP, template-independent polymerases readily incorporate the modified nucleotide at the 3’-end of DNA and RNA oligonucleotides. Overall, this article represents a first step towards the development of a universal, enzymatic GalNAc-labelling method for therapeutic oligonucleotides.