Pseudouridine and 5-methylcystidine Modified mRNA Targeting

Example 9

It was also tested whether 100% substitution of both uridine and cytosine with pseudouridine and 5-methylcystidine nucleotides would affect miRNA-dependent gene silencing. To that end, GFP or GFP-4x126TS mRNA were in vitro transcribed with 100% pseudouridine and 100% 5-methylcystidine and transfected into Ad-HEK293 cells that were priory (24 hr) transfected with miR-126 or miR-143 mimics. The inhibitory effect of miR-126 on the expression of the double-modified GFP-4x126TS mRNA were compared to the unmodified mRNA after 24 hr. Cells transfected with pseudouridine and 5-methylcystidine modified mRNA, miR-126 and not miR-143 reduced the percentage of GFP positive cells and inhibited GFP expression to the same extent as the unmodified mRNA (FIGS. 44A-44C). Thus, our data show that complete substitution of pseudouridine, or combination of pseudouridine and 5-methylcystidine modified mRNA, can still be targeted by microRNA-dependent silencing.