FIG. 5.

RNA binding assay. (A) Scheme of 3′UAR-PPMO-mediated competition with 5′UAR/3′UAR duplex formation. Two RNAs, one representing the 5′ terminal 190 nucleotides (5′RNA) and the other representing the 3′ terminal 111 nucleotides (3′RNA), could form 5′/3′RNA duplex when incubated together. Addition of 3′UAR-PPMO causes a reduction in the formation of 5′/3′RNA duplex, through binding to the 3′RNA. Sequences for both WT and mutant (MT) 5′RNA and 3′RNA are presented. Mutated nucleotides are indicated in shaded circles. (B) Competition assays. ³²P-labeled 3′RNA probe (lane 1) was converted to a 5′/3′RNA duplex when incubated with cold 5′RNA (lane 2). Indicated amounts of 3′UAR-PPMO were added to the mixture of 5′RNA and 3′RNA (lanes 3–6 and 9–12). The reactions were then analyzed on a nondenaturing polyacrylamide gel. The effect of 3′UAR-PPMO on 5′/3′RNA duplex formation was analyzed using WT or MT RNA. The positions of free probe of 3′RNA, 5′/3′RNAs duplex, and 3′RNA/3′UAR PPMO complex are indicated on the left side of the gels; the concentrations of 3′UAR-PPMO are indicated on top of the gel. (C) Quantification of WT and MT 5′/3′RNA duplexes with increasing amounts of 3′UAR-PPMO. The amount of 5′/3′RNA duplex remaining after competition treatment with 3′UAR-PPMO was measured using a PhosphoImager. For each 3′UAR-PPMO concentration, the value = amount of 5′/3′RNA duplex with PPMO/amount of 5′/3′RNA duplex without PPMO × 100. Mean values from three experiments are shown.