Radiolabeled Target RNA Synthesis

To generate 5′ and internally radiolabeled target RNAs, synthesized 5′ and 3′ fragments of the target RNA were ³²P-radiolabeled at the 5′ end using T4 polynucleotide kinase (Takara). After gel purification, the radiolabeled 5′ fragment was ligated to a 5′-phosphorylated unlabeled 3′ fragment, and the radiolabeled 3′ fragment was ligated to an unlabeled 5′ fragment by splinted ligation with T4 DNA ligase (NEB) at 30 °C for 2 h, respectively. The radiolabeled target RNA was gel-purified and used for the assay. BmAgo3 immunoprecipitation for the cleavage assay was described previously (Izumi et al, 2022 (link)). A target cleavage assay was performed at 40 °C for 2 h in a 10 μl reaction containing 3 μl of 40× reaction mix (Haley et al, 2003 (link)) and 2 nM ³²P-radiolabeled target RNA. After the supernatant was removed, the BmAgo3-bound beads were further incubated in buffer D containing 5 mM ATP and 350 nM recombinant proteins at 30 °C for 1 h. Then, the supernatant and bead fractions were treated separately with proteinase K, and the target RNA was purified by EtOH precipitation. An image of the target RNA, separated on an 8% denaturing polyacrylamide gel, was captured using an FLA-7000 imaging system (Fujifilm Life Sciences). The oligonucleotides used for target RNA preparation are listed in Table EV1.