Pseudouridine Impacts on miRNA-Mediated Gene Silencing

Example 8

To test whether modified nucleic acids affect the efficiency of miRNA-target site recognition and miRNA-dependent gene silencing, GFP or GFP4x126TS mRNA was in vitro transcribed with substitutions of uridine with pseudouridine (0%, 25%, 50% or 100%). Since ad-HEK293 cells do not express miR-126 or miR-143, we transfected with miR-126 mimic or miR-143 mimic 24 hr prior to transfection with GFP or GFP4x126TS mRNAs to ensure for miRNA/RISC assembly and GFP expression levels were assessed after 24 hr. In the control, unmodified (0% Pseudouridine) GFP transfected cells, the over-expression of miR-126-3p or miR-143 did not have any effect on GFP expression (FIGS. 43A-43C). This was also observed in cells transfected with GFP-mRNA containing increasing percentage of Pseudouridine (FIGS. 43A-43C). However, in cells transfected with GFP4x126TS mRNA, the over-expression of miR-126 and not miR-143 dramatically reduced the percentage of GFP positive cells and inhibited GFP expression. The miR-126-specific inhibition was not affected by increasing the percentage of pseudouridine substitution (FIGS. 43A-43C).