After incubation with cathepsin B, the DNA peak of ODN7UV disappeared, and instead, a new DNA peak at 5.86 min was observed which corresponds to Val-Ala-T12 (Figures and), demonstrating the enzymatic cleavage of ODN7UV by cathepsin B. E-64, a commercial protease inhibitor, was used to inhibit the activity of cathepsin B. As shown in Figure , 0.2 U/mL cathepsin B failed to efficiently cut 10 M ODN7UV in buffer A containing 1 M E-64 (purple line), suggesting again, cathepsin B is responsible for the enzymatic cleavage of the dipeptide in ODN7UV. To investigate if the released payloadphosphate was further converted to dephosphorylated compound by phosphatase, we conjugate 4-methylumbelliferone (4MU) with peptide. 4MU is a coumarin fluorescent dye, and its phosphate derivative is a commercial fluorescent probe for phosphatase enzymes. As shown in Figure Covalent conjugation of small molecules with Val-Ala(NB) linker through phosphoramidite chemistry is limited to those small molecules containing a hydroxy group. To expand the general utility of Val-Ala(NB) dipeptide linker in the facile synthesis of oligonucleotide-small molecule conjugates, we studied the solid-phase conjugation of Val-Ala(NB) linker with payloads containing an amino group via a carbamate linker. As shown in Figure , hydroxy group of Val-Ala(NB) was activated by N,N'-disuccinimidyl carbonate at 37 °C overnight, followed by reaction with the secondary amino group of Rucaparib. After washing, deprotection and purification, DNA-Val-Ala(NB)-