Extended Data Fig. 7: In vitro and cellular activities of pre-miR-155-RiboTAC and its derivatives.
From: Programming inactive RNA-binding small molecules into bioactive degraders
a, left: Representative gel autoradiogram of the cleavage of 5′-[32P]-pre-miR-155 by RNase L induced by pre-miR-155-RiboTAC and quantification thereof (IC50 = ~100 nM; n = 3 independent replicates); right: Representative gel autoradiogram of the pre-miR-155-RiboTAC-induced cleavage of pre-miR-155 as a function of pre-miR-155-binder concentration (IC50 = 1.9 ± 0.5 µM), demonstrating pre-miR-155-binder and pre-miR-155-RiboTAC bind the same site, and the corresponding quantification thereof (n = 3 independent replicates). b, Representative gel autoradiogram of (left to right): RNase L cleavage of cleavage site mutant of pre-miR-155 induced by pre-miR-155-RiboTAC; RNase L cleavage of the binding site mutant pre-miR-155 induced by pre-miR-155-RiboTAC; and RNase L cleavage of WT pre-miR-155 induced by Ac-RiboTAC, which lacks the RNA-binding module (n = 3 independent replicates). For panels a and b, “H” indicates a hydrolysis ladder while “T1” indicates treatment with RNase T1, which cleaves guanosine nucleotides. c, Effect of pre-miR-155-RiboTAC (n = 3 biological replicates) or pre-miR-155-Ctr (n = 4 biological replicates) on the viability of MDA-MB-231 cells following a 48 h treatment, as assessed by WST-1 assay. d, pre-miR-155-RiboTAC (single dose) reduces mature miR-155 levels in MDA-MB-231 cells in a time-dependent manner (n = 3 biological replicates). e, Duration of pre-miR-155-RiboTAC’s effect on pre-miR-155’s levels after a 48 h treatment (wash out experiment; n = 3 biological replicates). The compound-containing medium was removed after the treatment period, and total RNA was harvested at the indicated times post-treatment, affording a t1/2 = 19.3 h. The observed half-life is in accord with reported half-lives of miR-155, which range from 12.4–28 h15. f, Effect of Ac-RiboTAC (green), the RNase L recruiter lacking the RNA-binding module, and pre-miR-155-Ctr (red), a chimera with the less active regioisomer of the RNase L recruiter, on miR-155 biogenesis after a 48 h treatment as determined by RT-qPCR (n = 3 biological replicates). g, Effect of pre-miR-155-RiboTAC and pre-miR-155-binder on pre-miR-155 (left; n = 6 biological replicates) and mature miR-155 (right; n = 3 biological replicates for compound-treated cells; n = 6 biological replicates for vehicle-treated) levels in CFPAC-1 (pancreatic ductal adenocarcinoma) cells, as determined by RT-qPCR. h, Correlation of miRNome-wide changes of MDA-MB-231 cells treated with pre-miR-155-RiboTAC (100 nM) or LNA-155 (50 nM) (n = 3 biological replicates). Comparison of normalized read counts for each miRNA between pre-miR-155-RiboTAC and LNA-155 treatment (left). Effect of pre-miR-155-RiboTAC (100 nM, n = 3 biological replicates) on other miRNAs sharing the targeted A bulge (right). i, Correlation of transcriptome-wide changes of MDA-MB-231 cells treated with pre-miR-155-RiboTAC (100 nM) or LNA-155 (50 nM) (n = 3 biological replicates). From left to right: (i) Volcano plot of transcriptome-wide changes of MDA-MB-231 cells treated with pre-miR-155-RiboTAC (100 nM) vs. vehicle and LNA-155 (50 nM) vs. vehicle after a 48 h treatment period; (ii) comparison of normalized read counts for each gene between pre-miR-155-RiboTAC and LNA-155 treatment; (iii) comparison of normalized % change for genes commonly affected by both pre-miR-155-RiboTAC and LNA-155 treatment (n = 26 genes, right). All data are reported as the mean ± S.D. All p-values were calculated using a two-tailed Student’s t-test.
