FIGURE 2.
Determining the DNA overhang requirement for RNA 5′-adenylation with T4 DNA ligase and ATP. These assays used RNA subtrate S1 (5′-GGAAGAGAUGGCGACGG-3′) and were performed in 40 mM Tris (pH 7.8), 10 mM MgCl2, 10 mM DTT, and 500 μM ATP at 37°C (5 μM RNA; t = 0 h, 2 h, 8 h, and 24 h). The yields of 5′-AppRNA for DNA overhang lengths of 1 nt, 5 nt, and 10 nt were 8%, 26%, and 37% under these conditions, indicating that the longer 10-nt overhang is preferred. A 20-nt DNA overhang led to approximately the same 5′-adenylation yield as a 10-nt overhang (data not shown). Additional studies defined optimal incubation conditions that differ in several ways from those used in this particular experiment (see text).