Antisense oligonucleotides (ASOs) represent a potent and emerging class of therapeutics with the ability to modify gene expression. This potential makes them attractive therapies for various conditions, including rare hereditary disorders where limited treatment options exist.¹ ASOs recognize and bind specific RNA sequences, offering targeted intervention mechanisms. More broadly, oligonucleotide drugs (including ASOs) have garnered increasing interest, with 13 such therapeutics approved in the United States as of 2021.²

A significant challenge in the development of therapeutic ASOs is their limited ability to translocate membranes due to their polarity and size. However, recent studies have demonstrated that conjugating peptides or lipids to ASOs can improve their ability to cross membranes and also enhance their pharmacokinetic properties.^3,4,5 Additionally, the utilization of locked nucleic acids (LNAs) has been shown to enhance target affinity and reduce off-target effects. To the best of our knowledge, prior to the work described herein, LNA-containing ASOs have yet to be used in native chemical ligation (NCL)-based strategies to efficiently forge peptide-oligo constructs. Our research aims to enhance current ASO conjugation strategies by employing NCL to synthesize multifunctional peptide- and sugar-ASO conjugates. Access to cysteine bearing conjugates using ligation chemistry provides opportunities for further modifications such as fluorophore tagging and lipidation, amongst others, via alkylation of the cysteine thiol. Simple desulfurization also enables facile removal of the thiol handle. By expanding the application of NCL to ASOs containing LNA and phosphorothioate (PS) modifications, this synthetic methodology facilitates the creation of modular multifunctional conjugates, thereby broadening the scope of ASOs for application as probes and therapeutics.