Extended Data Fig. 10: A retrospective analysis of ‘failed’ CAGE-prox predictions and other potential expanded applications of CAGE-prox.

a, The CAGE-prox predicted mutations that have been experimentally evaluated as failed can be classified into two categories: ‘leaky’ (fail to block the protein activity with the inserted caged ONBY) and ‘dead’ (fail to restore the protein activity after photo-decaging). We calculated the frequency fold change (FC) of each native amino acid in each category. The fold change was defined as the frequency of a residue in a certain category/frequency in all predicted residues. x axis represents the fold change (expressed in log₂) and y axis represents the log(P value). As shown in the plot, ONBY insertions at native Gly and Ser positions are more likely to result in leaky mutants (left) whereas insertion at native Arg positions is more likely to result in dead mutants (right). As expected, a native Tyr position is less likely to fail. A total of 56 anchor residues from 7 different proteins was analysed using hypergeometric test. b, CAGE-prox-enabled control of auto-phosphorylation of Src kinase. HEK293T cells were co-transfected with Src-TAG mutants and the \({{\rm{ONBY\mbox{--}RS-tRNA}}}_{{\rm{CUA}}}^{\mathrm{pyl}}\) pair, and cultured for 24 h in the presence of ONBY. After UV-triggered decaging of the Src-OBNY variants, cells were cultured at 37 °C for another 3 h before the auto-phosphorylation level of each Src mutant was detected by immunoblotting. n = 2. c, Cell-specific targeting of a POI by CAGE-prox by adding the cancer-cell-targeting ligand to PA and the N-terminal domain of LF (LFn) to a POI, respectively. All above-mentioned samples are biological replicates.