Engineering Enzyme-Cleavable Oligonucleotides by Automated Solid-Phase Incorporation of Cathepsin B Sensitive Dipeptide Linkers

Oligonucleotides containing cleavable linkers have emerged as versatile tools to achieve stimulus-responsive and site-specific cleavage of DNA. However, the limitations of previously reported cleavable linkers including photolabile and disulfide linkers have restricted their applications in vivo. Inspired by the cathepsin B-sensitive dipeptide linkers in antibody-drug conjugates (ADCs) such as Adcetris, we have developed Val-Ala-02 and Val-Ala-Chalcone phosphoramidites for the automated synthesis of enzyme-cleavable oligonucleotides. Cathepsin B digests Val-Ala-02 and Val-Ala-Chalcone linkers efficiently, enabling cleavage of oligonucleotides into two components or release of small-molecule payloads. Based on the prior success of dipeptide linkers in ADCs, we believe that these dipeptide linker phosphoramidites will promote new clinical applications of therapeutic oligonucleotides.

Automated summary

Oligonucleotides containing cleavable linkers are versatile tools for stimulus-responsive and site-specific DNA cleavage, but their previous limitations have restricted their in vivo applications. Inspired by cathepsin B-sensitive dipeptide linkers in ADCs, we have developed phosphoramidites of Val-Ala-02 and Val-Ala-Chalcone for automated synthesis of enzyme-cleavable oligonucleotides. Cathepsin B efficiently digests these linkers, enabling cleavage of oligonucleotides or release of small-molecule payloads. We believe that these dipeptide linker phosphoramidites will expand the clinical applications of therapeutic oligonucleotides.