Figure 7

Biotin RISC differs only slightly from native RISC in affinity for centered sites. (A) Schematic diagram showing the bio-layer interferometry (BLI) experimental design. Cells were either transfected with biotinylated miR-182-5p or induced to over-express miR-182-5p, and RISC complexes containing these miRNAs were affinity purified. The two RISC solutions were used in parallel BLI runs to detect association and disassociation with RNA oligos containing the binding sites. The arrows indicate the sequential immersion of the sensors in a row. Lane 1, no lysate; lane 2, no oligo; lane 3, miR-182-5p 3 prime binding site; lane 4, miR-182-5p imperfect centered site with GU wobble; lane 5, miR-182-5p imperfect centered site; lane 6, miR-182-5p perfect centered site; lane 7, miR-182-5p seed site. (B) The binding between miR-182-5p and the RNA oligo is illustrated. The miRNA is depicted on the top strand, and the binding site in the oligo on the bottom strand. Perfect Watson-Crick matches are indicated with the vertical line, and GU wobble sites by a colon. (C) A representative association/disassociation curve from the BLI showing the wavelength interference (nm) versus time. The dashed line indicates the time at which the BLI sensors were removed from the RISC solution and transferred to buffer. (D) Rate constants for association were calculated independently from four biological replicates, and plotted separated for biotin RISC (B) and native RISC (N) across all five binding sites. Error bars represent standard error of the mean (sem). (E) Rate constants for disassociation were calculated and plotted as for (D). Error bars represent standard error of the mean (sem). (F) The normalized affinity for each site type was calculated by dividing the association rate constant by the disassociation rate constant, and normalizing all site types to the ratio for the seed site. This shows that, with the exception of 3' sites, the relative affinities between biotin RISC and native RISC are essentially equivalent.