Electrophoretic mobility shift assay (EMSA)

The sequences of the different nucleic acids used in this study are tabulated in Table 2. The synthesized RNA strands were purchased from ST Pharm (Republic of Korea). The 5′-ends of the RNA and DNA molecules were labeled with [γ-³²P]-ATP using T4 polynucleotide kinase (Roche, Germany). To remove unincorporated [γ-³²P]-ATP, the mixture was desalted using an RNase-free Sephadex G-25 column (GE Healthcare, Sweden). The labeled probes were then incubated with ZmASCH for 30 min at 310 K in a buffer solution consisting of 20 mM Tris–HCl (pH 7.5), 10 mM magnesium acetate, 300 mM potassium chloride, 100 ng/μL bovine serum albumin, and 100 ng/μL heparin. The mixtures were loaded onto 15 or 20% (w/v) non-denaturing polyacrylamide gel (40:1). Electrophoresis was conducted at 70 V for 60 min at 298 K in Tris-borate-EDTA buffer (89 mM Tris, 89 mM boric acid, and 2 mM EDTA). The results were visualized by using a Fuji phosphorimager.

Oligonucleotides used in this study.

The methylated nucleotide is indicated with “m” in the parenthesis, while the theoretical cleavage sites of ssRNA (17mer)s are indicated with “^∨”.