Synthetic Design of Asymmetric miRNA with an Engineered 3′ Overhang to Improve Strand Selection

We developed a novel miRNA design that significantly improves strand selection within the RISC complex by engineering the 3′ end by adding extra nucleotides. Addition of seven nucleotides at the 3′ ends of the miR or miR* strand resulted in a thermodynamic asymmetry at either of the two ends, which resulted in selective RISC recruitment, as demonstrated by a stem-loop PCR experiment. Such selective recruitment was also corroborated at the protein level by western blot analysis. To investigate the functional effect because of selective recruitment, we performed apoptosis and metastasis studies using human colon carcinoma cells (HCT116) and human osteosarcoma cells (MG63). These experiments indicated that recruitment of the miR strand is responsible for inducing apoptosis and inhibiting the invasiveness of cancer cells. Recruitment of the miR* strand, on the other hand, had the opposite effect. To the best of our knowledge, our strand engineering strategy is the first report of improved strand selection of a desired miRNA strand by RISC without using any chemical modifications or mismatches. We believe that such structural modifications of miR34a could mitigate some of the off-target effects of miRNA therapy and would also allow a better understanding of sequence-specific gene regulation. Such a design could also be adapted to other miRNAs to enhance their therapeutic potential.