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. 2020 May 28;7(13):1903451. doi: 10.1002/advs.201903451

Figure 9.

Figure 9

The mode of action for the optimal small molecule OC‐3. a) Left: Docking pose 1 shows OC‐3 (oxygen atoms in red, hydrogen atoms in white, carbon atoms in green) bound to the loop formed by miR‐214 (backbone: green ribbons) and TRAF3 mRNA (backbone: orange ribbons) in the complex of AGO protein (grey ribbons). Docking pose 1 has the highest score among the four obtained docking poses. Right: A partial magnification of the docking pose 1 illustrates the binding sites between the small molecule OC‐3 and loop formed by miR‐214 and TRAF3. Potential hydrogen‐bonding interactions between the small molecule OC‐3 and G13 and C10 base of the loop are highlighted (green dashed lines). The other three computational docking poses showing OC‐3 bound to the loop formed by miR‐214 and TRAF3 mRNA in the complex of AGO protein. b) Pull‐down assay using streptavidin magnetic bead to examine the interaction between biotin‐labeled loop (formed by miR‐214 and mutated TRAF3 mRNA) and the small molecule OC‐3. Small molecule OC‐3 captured by the mutated loop was examined by LC‐MS. c) TRAP staining (left) and real‐time PCR analysis of CTSK mRNA level (right) in RAW 264.7 cells after treatment with vehicle, small molecule OC‐3, Loop, G13 mutated loop, OC‐3 + loop, and OC‐3 + G13 mutated loop, respectively. d) Pull‐down assay using streptavidin magnetic bead to examine the interaction between biotin‐labeled loop (formed by mutated miR‐214 and TRAF3 mRNA) and the small molecule OC‐3. Small molecule OC‐3 captured by the mutated loop was examined by LC‐MS. e) TRAP staining (left) and real‐time PCR analysis of CTSK mRNA level in RAW 264.7 cells after treatment with vehicle, small molecule OC‐3, Loop, C10 mutated loop, OC‐3 + loop, and OC‐3 + C10 mutated loop, respectively. f) Pull‐down assay using streptavidin to examine the interaction between biotin‐miR‐214‐TRAF3 loop and the methylated small molecule OC‐3. Methylated small molecule OC‐3 captured by the loop was examined by LC‐MS. g) TRAP staining (left) and real‐time PCR analysis of CTSK mRNA level in RAW 264.7 cells after treatment with vehicle, small molecule OC‐3, and methylated small molecule OC‐3, respectively. *p < 0.05, **p < 0.01, ***p < 0.001 versus vehicle group.