Towards the Enzymatic Labeling of Oligonucleotides With Triantennary N ‐Acetyl Galactosamine

The introduction of chemical modifications into oligonucleotides enhances backbone stability against nuclease degradation and improves biological activity. Although achieving cell-, tissue-, and organ-specific delivery remains challenging, conjugation to triantennary N-acetylgalactosamine (triGalNAc) ligands enables receptor-mediated delivery of nucleic acids to hepatocytes. TriGalNAc ligands are usually attached to the termini of therapeutic oligonucleotides via labor-intensive methods that require specialized equipment and expertise. As an alternative, we explored enzymatic labeling of oligonucleotides with triantennary GalNAc ligands, providing a strategy that is compatible with any sequence and does not require complex chemical synthesis. Two complementary strategies for triGalNAc labeling were employed. In the first, we modified the γ-phosphate of ATP with a triGalNAc moiety and tested its kinase-catalyzed transfer to the 5' termini of oligonucleotides. In the second, we functionalized the 3' position of an LNA nucleoside triphosphate with a triGalNAc residue and used this analog for template-independent 3'-end labeling of oligonucleotides. While kinases do not seem to fully 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 work represents a first step towards the development of a universal, enzymatic triGalNAc-labeling method for therapeutic oligonucleotides.