Initially, we tested three variants of an ASO sequence we had previously identified that targets the mRNA for mVenus (mV), a yellow fluorescent protein. Each variant contained three thymines (dTs) replaced with amino-C6-dTs at different positions. The three amino-ASOs were reacted with a biotinylated 2-nitrobenzyl N-hydroxysuccinimide (NHS)-ester photocleavable group (uvLA-biotin, Fig. ) and purified by HPLC (see Supplementay Information), prior to binding either monovalent streptavidin (mSA) or wild-type streptavidin (tetSA) to sterically block RNase H from binding the hybrid DNA:RNA duplex. These UV light-activatable (uvLA) ASOs were tested against mV-mRNA with or without illumination (Supplementary Fig. ) and analysed by agarose gel electrophoresis. A better 'off-state' (least mRNA degradation) was observed when the modified dTs were distributed throughout the entire sequence in ASO uvLA-V1, rather than concentrated at the terminus. Moreover, when bound to mSA rather than tetSA, the light-sensitive moieties appeared to photocleave more efficiently upon UV irradiation, resulting in a better ON/OFF ratio. Thus, we continued using mSA in the subsequent experiments.