One-step and two-step syntheses of YBL059W lariat RNA. (A) Attempting one-step synthesis according to the strategy of Figure 1A using the 7S11 deoxyribozyme, which provides an undetectable (<1%) amount of lariat RNA (12% PAGE). The lariat standard (lar) was available from the two-step route of B. t = 0, 1.5, and 3 h. Inclusion of a disruptor DNA oligonucleotide (Wang and Silverman 2005a) did not increase the yield of lariat (data not shown). (B) Two-step synthesis according to the strategy of Figure 1B, which proceeds efficiently for both steps (12% PAGE). t = 0, 0.5, and 1.5 h for branched RNA synthesis using the 7S11 deoxyribozyme, and t = 2 and 10 min for lariat synthesis using T4 RNA ligase (– denotes PAGE-purified branched RNA incubated without T4 RNA ligase). As shown in Figure 1B, L denotes the left-hand RNA substrate, which contains the 32P-radiolabel.
