Fla 7000 imaging system

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.

ATP hydrolysis reaction was performed at 25 °C for 1 h using 2 μg of recombinant protein in buffer D containing 0.02% Triton X-100, 2 μM 36-nt polyU RNAs, and 0.23 μl of [γ-³²P] ATP (6000 Ci/mmol, Perkin Elmer). Twenty percent of the reaction mixture was spotted onto a polyethyleneimine cellulose plate (MACHEREY-NAGEL) that had been pre-run with water for 2 h. The plate was then run with 450 mM ammonium sulfate for 1 h, dried, and analyzed using an FLA-7000 imaging system (Fujifilm Life Sciences).

To describe TRF2 binding to telomeric DNA, electrophoresis in 5% non-denaturing polyacrylamide in 0.25×Tris-Borate-EDTA (TBE) buffer was used. Reactions containing the same amount of fluorescently labeled DNA (3 pmol) and increasing amounts of protein TRF2 (3–15 pmol) were prepared. DNA was labeled with fluorophore Alexa Fluor 488. The influence of protein Rap1 on DNA-binding affinity of TRF2 was detected in reaction mixtures composed of a constant amount of labeled DNA (5 pmol), a fixed amount of protein TRF2 (10 pmol) and increasing amounts of protein Rap1 (20–80 pmol). In both cases, the reactions were supplemented with buffer (50 mM NaCl, 50 mM sodium phosphate, pH 7.0) to a volume of 15 and 3 μl of 6× loading buffer (60% glycerol; 10 mM Tris/HCl, pH 7.6 and 60 mM ethylenediaminetetraacetic acid). Reaction mixtures were incubated for 15 min on ice and then loaded on horizontal 5% (w/v) non-denaturing polyacrylamide gels in 0.25×TBE buffer. The electrophoresis proceeded at 1 V/cm for 45 min and for an additional 3 h at 2 V/cm at 4°C. Fluorescently labeled DNA in gels were analyzed with a FLA 7000 imaging system (Fujifilm).