The two ASOs chosen from these screens for orthogonal targeting of mC (C1) and mV (V2), both contained four amino-C6-dT modifications. They were modified with bLA-biotin and uvLA-biotin (Fig. ) respectively, purified by HPLC (see Supplementary Information) and bound to mSA (forming bLA-C1 and uvLA-V2). mV-and mC-mRNA were incubated with the two photocaged ASOs and RNase H, to test light-controlled mRNA degradation (Fig. ). In the absence of irradiation, both mRNAs remained intact. Strikingly, upon illumination with blue light, mC-mRNA was degraded while mV-mRNA stayed intact. Similarly, mV-mRNA was degraded selectively after irradiation with UV light, with no degradation being observed for the mC-mRNA. We then used these orthogonal LA-ASOs to selectively control the cellfree protein synthesis of the two different proteins (Fig. ). The DNA templates of mV and mC, bLA-C1, uvLA-V2, and RNase H were added to the CFPS system, incubated for 4 hours following different patterns of illumination, and the fluorescence of both proteins was measured. Without illumination, both proteins were expressed to similar levels observed in the absence of ASOs (17% increase for mC and 23% decrease for mV, both non-significant). Upon UV irradiation, uvLA-V2 selectively knocked down mV fluorescence by 56%, whereas mC was only reduced by 16% (non-significant, p-value = 0.129). Vice versa, upon blue light irradiation, bLA-C1 selectively knocked down mC fluorescence by 62%, with a 30% reduction observed for mV fluorescence (significant against positive control, p-value = 0.032). Excitingly, this demonstrated we were able to control gene knockdown in CFPS using two orthogonally light-controlled ASOs.