Extended Data Fig. 6: pre-miR-155-binder and derivatives directly engage pre-miR-155 and other A bulge containing miRNA precursors in MDA-MB-231 cells and in vitro.
From: Programming inactive RNA-binding small molecules into bioactive degraders
a, The Chem-CLIP probe for target validation studies (pre-miR-155-Chem-CLIP) was synthesized by conjugating pre-miR-155-COOH to chlorambucil (CA; cross-linking module; blue triangle) and biotin (pull-down module; yellow circle). A control Chem-CLIP probe, Ac-CA-biotin, comprises the cross-linking and pull-down module but lacks the RNA-binding module and accounts for non-selective reaction of the CA module. b, Chem-CLIP is a proximity-based reaction driven by the RNA-binding module. Following treatment, total RNA is isolated and purified with streptavidin beads and analysed by RT-qPCR. c, Results of target validation studies in MDA-MB-231 breast cancer cells. Cells were treated with either Ac-CA-Biotin (100 nM) or pre-miR-155-Chem-CLIP (100 nM). Enrichment of pre-miR-155 in the pulled-down fraction, as compared to the starting cell lysate, was quantified by RT-qPCR (left). Co-treatment with pre-miR-155-Chem-CLIP and pre-miR-155-binder resulted in dose dependent depletion of pre-miR-155 in the pull-down fraction, indicating that the Chem-CLIP probe and the simple binding compound pre-miR-155-binder occupy the same site (n = 3 biological replicates). d, pre-miR-155-Chem-CLIP cross-links to pre-miR-155 at a nucleotide 5 bp from the A bulge binding site (indicated with a blue arrow in the secondary structure), identified by in vitro Chem-CLIP-Map-Seq. e, Effect of pre-miR-155-binder (red; left) and pre-miR-155-amide-binder (blue; middle) (n = 3 biological replicates) on pri-, pre-, and mature miR-155 levels as well as the two compounds’ effects on cellular viability (right) (n = 4 biological replicates). f, Chem-CLIP analysis (100 nM pre-miR-155-Chem-CLIP) of miRNA precursors that have the same bulge as pre-miR-155 bound by pre-miR-155-binder (n = 3 biological replicates) where ND indicates that the miRNA was not detectable in the pulled down fraction. Effect of pre-miR-155-amide-binder (0.1 to 10 μM) on the levels of miRNAs that contain the A bulge in miR-155, as determined by RT-qPCR (n = 3 biological replicates). Pre-miR-155-RiboTAC (100 nM) has no effect on the levels of mature RNAs that share C1’s A bulge in miR-155 (Extended Data Fig. 6h). All data are reported as the mean ± S.D. All p-values were calculated using a two-tailed Student’s t-test.
