FIGURE 7.

Effects of 3′-capping of the DNA blocking oligo and RNA substrate. (A) 3′-capping of the DNA blocking oligo suppresses formation of the blocking oligo ligation side product, but at the expense of adenylation rate. RNA substrate S1 was reacted with the 3′-uncapped (3′-OH), 2′,3′-dideoxy-capped (3′-dd), or 3′-phosphate-capped (3′-p) +4/0 blocking oligo, ATP, and T4 RNA ligase as in Figure 3 ▶. The RNA substrate was either 2′,3′-diol or 2′,3′-cyclic phosphate as shown. (B) Including a 2′,3′-cyclic phosphate on the RNA substrate prevents the circularization and oligomerization side reactions but not blocking oligo ligation. The DNA blocking oligo was 3′-uncapped. For 5′-adenylation of RNA substrate S1 that has no 2′,3′-cyclic phosphate in the absence of a DNA blocking oligo, see the far left of Figure 3 ▶. The faint set of bands in the middle of the final time point for the fifth set of lanes is of unknown origin. For all sets of lanes in both panels, the four time points are at 0.5 min, 10 min, 1 h, and 6 h (20% PAGE); the fifth time point in A for the final two sets of lanes is at 15 h. The sets of lanes are numbered at the bottom for reference (see text). For the experiments in sets of lanes 4 and 5, the 5′-AppRNA yields at 6 h were 60% and 50%, respectively; the yields at 15 h were 80% and 70%. Note that RNA with a 2′,3′-cyclic phosphate has a slightly faster mobility than 2′,3′-diol RNA due to the additional negative charge.