Extended Data Fig. 11: In vitro and cellular characterization of c-Myc-binder and its derivatives.

a, Representative binding curves to measure the affinity of c-Myc-binder, c-Myc-RiboTAC and c-Myc-Ctr for a Cy5-labelled model of the c-Myc IRES 5’UUCG/3’ACCC 2×2 internal loop or a fully paired RNA control (n = 2 independent experiments). b, Chemical structures of c-Myc-Chem-CLIP and Ctr-Chem-CLIP probes (top) and target validation studies in HeLa cells (bottom). c-Myc-Chem-CLIP dose dependently enriches MYC mRNA (lower left; n = 3 biological replicates), which is competed by c-Myc-binder (lower right; n = 6 biological replicates). c, Effect of c-Myc-binder on MYC mRNA and protein levels in HeLa cells (n = 3 biological replicates). d, c-Myc-RiboTAC induces in vitro cleavage of the WT MYC IRES (left), while no cleavage of a mutated base pair control was observed (middle). Pre-miR-155-RiboTAC and c-Myc-RiboTAC have no effect on the non-cognate RNA (right) (n = 3 biological replicates for all panels). Concentrations were selected based on the cellular activity for the cognate target. e, Chemical structures of c-Myc-amide-binder and Ac-RiboTAC. f, Effect of c-Myc-amide-binder, Ac-RiboTAC, and c-Myc-Ctr on MYC mRNA and protein levels in HeLa cells (n = 3 biological replicates). g, Effect of siRNA knock-down of RNase L (~85%) on the activity of c-Myc-RiboTAC in HeLa cells (n = 3 biological replicates). h, Effects of c-Myc-RiboTAC and BRD4 degrader MZ1 on the proliferation and apoptosis of HeLa cells upon 48 h treatment (n = 3 biological replicates). i, Effect of c-Myc-RiboTAC on MYC mRNA levels (left; n = 3 biological replicates), protein abundance (middle; (n = 2 biological replicates for vehicle (note that the third lane was excluded from quantification due to air bubble) and n = 3 biological replicates for compound-treated sampled), and proliferation (right; n = 3 biological replicates) in MDA-MB-231 cells. j, Effect of siRNA knockdown of RNase L on c-Myc-RiboTAC-mediated degradation of MYC mRNA in MDA-MB-231 cells (n = 3 biological replicates). k, Effect of c-Myc-RiboTAC on a c-Myc-IRES-luciferase reporter or a binding site mutant thereof in transfected HEK293T cells (n = 4 biological replicates). l, Transcriptome-wide changes observed in HeLa cells treated with vehicle, c-Myc-RiboTAC, scrambled siRNA, or MYC siRNA (n = 3 biological replicates). Volcano plots of transcriptome-wide changes of HeLa cells treated with MYC-siRNA (1 nM) vs. scrambled siRNA (1 nM) after a 48 h treatment period (left). Cumulative curves of MYC targets and HIF-1α targets in HeLa cells treated with c-Myc-RiboTAC (10 μM) vs. vehicle (middle) or c-Myc-siRNA (1 nM) vs. scrambled siRNA (1 nM) (right). m, Volcano plots of proteome-wide changes of HeLa cells treated with c-Myc-RiboTAC (10 μM) vs. vehicle (left) or MYC-siRNA (1 nM) vs. scrambled siRNA (1 nM) (right) after a 48 h treatment period (n = 3 biological replicates). n, Abundance of RNase L mRNA in various cell lines (left; n = 3 biological replicates). Effect of c-Myc-Ctr (10 μM) and c-Myc-RiboTAC (10 μM) on MYC mRNA levels in lymphoma or leukaemia cells, Raji, and HL-60 cells, respectively (middle left; n = 6 biological replicates for vehicle and n = 3 biological replicates for compound treatment) and on apoptosis (middle right and right; n = 2 biological replicates). Interestingly, these data suggest that RiboTAC activity is correlated with RNase L expression. All data are reported as the mean ± S.D. of biologically independent replicates. Statistical significance was calculated using a two-tailed Student’s t-test (b-h, n), Wald’s test (i, left and m), or Kolmogorov–Smirnov test (i, middle and right).

Source data