FIGURE 1.

Sequences 1-5 and the overall process for 3′-radiolabeling (using nucleotide transformation 20→21), and 5′-phosphorylation of the corresponding oligonucleotides (A); and 20% denaturing PAGE of oligonucleotides 1″-3″ in the presence of Xrn-1 in the absence (B) and presence of markers 4 and 5 (C). The difference between lanes 1-3 and 4-6 in (B) represents a 10-fold dilution in [Xrn-1], respectively, where A = 0.12 pg, B = 0.012 pg, or (−) no enzyme added, per experiment. Reactions were carried out in buffered solutions (100 mM NaCl, 50 mM Tris▪HCl, 10 mM MgCl₂, 1 mM DTT, pH 7.9) by incubating for 2 h (rt). Further characterization of the fragment of interest was obtained via MALDI-TOF, which matched the mass of ON 4 phosphorylated at the 5′-end 4 (D).