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. Author manuscript; available in PMC: 2021 May 13.
Published in final edited form as: J Am Chem Soc. 2020 May 4;142(19):8706–8727. doi: 10.1021/jacs.0c00768

Figure 5.

Figure 5.

Target profiling in cells using Chemical Cross-Linking and Isolation by Pull-down (Chem-CLIP). (A) Scheme of Chem-CLIP to react with an RNA target in a cell to validate target engagement of small molecules. (B) The compounds used in Chem-CLIP and competitive Chem-CLIP (C-Chem-CLIP). (C) The Chem-CLIP probe of bioactive ligand 9 (compound 11) reacts with tau pre-mRNA in LAN5 cells, whereas control probe 12 does not. C-Chem-CLIP shows that when increasing concentrations of 9 are preincubated with LAN5 cells, the pull-down of tau pre-mRNA with a constant concentration of 11 is diminished in a dose-dependent manner. (D) Compound 11 reacts with tau pre-mRNA in WT and DDPAC mini-gene-transfected HeLa cells, whereas 12 does not. Preincubation of 9 diminished pull-down of 11 in C-Chem-CLIP. Compound 11 does not pull-down I17T mutant tau pre-mRNA in transfected cells. Collectively, Chem-CLIP pull-down of 11 and C-Chem-CLIP studies show that target engagement is dependent on the presence of the 3D structure of the RNA target, as mutations abate pull-down. Error bars indicate SD. ** P < 0.01 and *** P < 0.001, as determined by a two-tailed Student t-test.