FIGURE 3.

Tagging RNA by splint-dependent ligation. (A) 0.5 µM chemically synthesized, radiolabeled RNA N₂₂ was ligated with 0.75 µM of different preannealed splint–donor duplex DNA combinations as indicated in the scheme at the top. Two different splints were used having a 6-nucleotide (nt) or 4-nt complementarity to the RNA 3′ end as indicated (splint 4 nt, splint 6 nt) (Table 1). Ligations were performed at two different temperatures for the times indicated. Reactions where stopped by mixing with 20 mM EDTA in formamide, and samples were analyzed by denaturing polyacrylamide gel electrophoresis. (B) The ligation reaction was carried out as in A with an enzymatically synthesized, radiolabeled RNA consisting of two species, 50 and 51 nt long. One splint (6 nt) (Table 1) had a 6-nt complementarity to the expected end of the 50-nt RNA, immediately followed by the sequence matching the tagging oligonucleotide. The second splint (6 ntN) (Table 1) had the same complementarity, followed by a position containing a mixture of all four nucleotides and then the sequence matching the tagging oligonucleotide. Ligations were carried out with either splint or the mixture of both as indicated. DNA marker sizes are indicated on the right.