A versatile strategy for oligonucleotide functionalization via on-support phosphitylation.

Chemically modified nucleic acids such as antisense oligonucleotides, small interfering RNA and CRISPR guide RNA have emerged as transformative modalities in modern medicine, providing new strategies for the treatment of genetic and metabolic disorders, viral infections and cancer. However, their application is limited by challenges related to intracellular delivery and targeting, advances in which would benefit from a broader range of methods for chemical functionalization. Here we describe a streamlined approach using inexpensive, bench-stable alcohols or nucleosides that enables site-selective modification at the 5' and 3' termini and at internal sites of synthetic oligonucleotides. Our strategy offers a conceptually straightforward solution to this problem through the in situ generation of phosphoramidites on the solid-supported oligonucleotide. The methodology displays high coupling efficiency, high functional-group compatibility with azides, amines and thiols, and efficient recovery of excess alcohols/monomers, thus offering a robust platform for advancing and diversifying therapeutic oligonucleotide modification.