Figure 1

Optimization of miR inhibitor efficiency. (a) miR inhibitor designs. Black line indicates the specific inhibitor sequence that is complementary to the full-length miR. Blue line indicates the double-stranded (ds) and stem loop RNA. (b) Predication of RNA secondary structure of the final inhibitor design by RNA mfold. The miR antisense sequence is boxed. (c) Structure of the vector construct used to transduce cells, either one or multiple miR inhibitors can be cloned into the vector. (d) Effects of secondary structure length, local AU content and last A ribonucleotide on miR inhibitor efficiency. The psiCHECK2 vector (Ck2) does not contain a miR-17 binding site after Rluc. The Ch2-miR-17 reporter contains a single miR-17 binding site cloned after Rluc. All co-transfections with inhibitors use the Ck2-miR-17 reporter. Normalized Rluc to Fluc ratio of miR reporter vector without miR-17 binding site was set as 100%. miR inhibitor designs were co-transfected with miR reporter containing a miR-17 binding site into HEK 293FT cells, which endogenously express miR-17. Rluc and Fluc activity was measured 48 h after transfection. The sponge plasmid has six tandem miR-17 binding sites. T-test was performed against the sample, marked by an arrow. *P<0.05 and **P<0.01. CMV, cytomegalovirus; EGFP, enhanced green fluorescent protein; HSV, herpes simplex virus.