To produce a better ON/OFF ratio, we proceeded to substitute one further dT with amino-C6-dT, for a total of four modifications (Fig. ). In addition, we screened for a new ASO targeting the same mRNA region, but with a higher potency and thymidines denser and more symmetrically distributed in the sequence (Fig. [mV220 vs mV229] and Supplementary Fig. ). Both the previous and the newly designed sequences containing four amino-C6-dTs (V2 and V3 respectively) were then modified with uvLA-biotin and caged with mSA (Fig. ). As expected, the addition of a fourth amino-dT resulted in a negligible decrease in ASO activity, while a fourth photocleavable streptavidin moiety visibly improved the 'on/off-state' (Fig. ). As modified ASO uvLA-V3 showed the best ON/OFF ratio, this was selected for further experiments. To control in vitro transcription (IVT) a linear DNA template encoding mVenus was transcribed in the presence of no ASO, V3, or uvLA-V3, and RNase H (Supplementary Fig. ). Without illumination, similar levels of mRNA were transcribed when using uvLA-V3 as if no ASO was present. Whereas when IVT took place following illumination, mRNA degradation was observed to similar levels of V3 amino-ASO (representing 100% photocleavage). We then moved to control cell-free protein synthesis with the uvLA-V3 ASO, using a commercial CFPS kit (PURExpress). V3 or uvLA-V3, and the mV DNA template were added to the CFPS system (supplemented with RNase H), and the yield of intact mVenus was measured by fluorescence after 4 hours (Fig. ). UV light irradiation in the absence of an ASO only resulted in a minor, non-significant decrease (p-value = 0.23) in protein synthesis levels, showing little/no UV-damage. The amino-ASO V3 decreased mV synthesis by 89% compared to the no ASO control, with no significant difference following illumination (p-value = 0.265). In the absence of UV light, uvLA-V3 only decreases protein synthesis by ~15% (nonsignificant, p-value = 0.084), whereas when irradiated with UV for 5 minutes, protein synthesis was reduced by ~75% compared to the no ASO control, and 84% of ASO activity was recovered when compared to the amino-ASO V3. These results demonstrate we were able to tightly control gene knockdown of CFPS using our light-activated ASOs.