Figure 4.

Ribonuclease T2 digestion to determine regiospecificity of bond formation. Lane 2 is the RNase T2 digestion product of the ligation in Fig. 1c (see Methods). Lane 1 is the RNase T2 digestion product of a ligated RNA of identical sequence, but with a labeled 3′,5′ linkage generated by T4 DNA ligase and a complementary DNA splint (42). Digestion products were analyzed on a 20% denaturing acrylamide gel. Lanes 3 and 4 are a time course of alkali hydrolysis at 5 and 10 min, respectively, of the ligated product digested in lane 1.