Extended Data Fig. 10: In vitro and in cellular characterization of c-Jun-binder and its derivatives. | Nature

Extended Data Fig. 10: In vitro and in cellular characterization of c-Jun-binder and its derivatives.

From: Programming inactive RNA-binding small molecules into bioactive degraders

Extended Data Fig. 10

a, Representative binding curves to measure the affinity of c-Jun-binder, c-Jun-RiboTAC, and c-Jun-Ctr for a model of the c-Jun RNA where the A bulge has been replaced with the fluorescent adenine mimic, 2-aminopurine (2AP) (n = 2 independent experiments). b, Representative binding curves to measure the affinity of c-Jun-binder, c-Jun-RiboTAC and c-Jun-Ctr for a Cy5-labelled model of the c-Jun IRES and a fully paired RNA control (n = 2 independent experiments). The fully paired RNA was created by mutating the A bulge binding site to an AU pair. c, Chemical structure of c-Jun-Chem-CLIP probe and control probe. Pull-down of the c-Jun mRNA in vitro by c-Jun-Chem-CLIP or Ctr-Chem-CLIP (n = 3 replicates). d, left: Target engagement studies completed by Chem-CLIP in MIA PaCa-2 cells (n = 3 biological replicates). Quantification of the enrichment of c-Jun mRNA by c-Jun-Chem-CLIP or Ctr-Chem-CLIP in pulled-down fraction as compared to RNA not subjected to pull-down; right: Competitive Chem-CLIP experiments in which MIA PaCa-2 cells were co-treated with c-Jun-Chem-CLIP at a constant concentration and increasing concentrations of c-Jun-binder, which depletes c-Jun mRNA in the pull-down fractions in a dose dependent fashion (n = 4 biological replicates). e, Effect of c-Jun-binder on c-JUN protein levels in Mia-PaCa-2 cells (n = 3 biological replicates). f, c-Jun-RiboTAC induces in vitro RNA cleavage with WT c-Jun construct (left), while no cleavage of the mutated base pair control (right) was observed (n = 3). All data are reported as the mean ± S.D. All p-values were calculated using a two-tailed Student’s t-test.

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