General Platform for Efficient and Modular Assembly of GalNAc-siRNA Conjugates via Primary Amines and o-Nitrobenzyl Alcohol Cyclization Photoclick Chemistry Enabling Rapid Access to Therapeutic Oligonucleotides.

Abstract

Oligonucleotide-based therapies, especially ligand-conjugated siRNAs, offer significant therapeutic potential for a wide array of diseases. However, conventional solid-phase synthesis and current postsynthetic in-solution conjugation methods face notable challenges related to efficiency, accessibility, and the scalability of diverse ligand-oligonucleotide conjugates. Herein, we introduce a novel strategy for highly efficient, rapid, and modular assembly of GalNAc-siRNA conjugates based on light-induced primary amine and o-nitrobenzyl alcohol cyclization (PANAC) chemistry. Leveraging the advantages of PANAC photoclick chemistry and modular conjugation linkers, our method enables the direct assembly of trivalent GalNAc (tGalNAc) with commercially available primary-amine-modified siRNAs. This approach demonstrates the efficient and rapid assembly of therapeutically relevant oligonucleotides with ligands of interest, offering operational simplicity and practicality; thus, it effectively overcomes the limitations of existing methods. More importantly, the developed siRNA-tGalNAc conjugates showed a robust gene silencing effect superior to the parent siRNA conjugate, highlighting the effectiveness of our method in generating and screening siRNA conjugates to enhance in vivo potency. Overall, our method enables modular and rapid assembly of therapeutically relevant oligonucleotide-tGalNAc conjugates using readily accessible oligonucleotides and commercially available tGalNAc-amine ligands. This approach expands the toolkit for generating ligand-oligonucleotide conjugates, providing a general and efficient platform with broad applicability, thereby advancing the optimization and development of oligonucleotide-based therapeutics.