Establishing generality of the RNA 5′-adenylation strategy using RNA substrates S1–S9 and optimal reaction conditions of 40 mM MOPS (pH 7.0), 10 mM MgCl2, 10 mM DTT, and 10 mM ATP at 25°C (1 μM RNA; t = 0 h, 8 h, and 24 h). For S9, the full complement leaves a blunt RNA 3′-terminus, whereas the partial complement leaves 17 nt of RNA unpaired at the 3′-terminus. See Materials and Methods for RNA sequences. The gel images have been aligned vertically solely for presentation purposes. Unlike in the experiments of Figure 2, which were not performed under these optimal reaction conditions, essentially no side products were observed here (<2%; data not shown). We attribute this largely to the elevated ATP concentration (Nilsson et al. 2001).
