Nature research paper: Structure of the OMEGA nickase IsrB in complex with ωRNA and target DNA
-thiogalactopyranoside and incubation at 18 °C for 20 h. Thecells were resuspended in buffer A , lysed by sonication and then centrifuged. The supernatant was mixed with Ni-NTA Agarose . The protein-bound column was washed with buffer A, buffer B and buffer C . The protein was eluted with buffer D . The cognate ωRNA of DtIsrB was transcribed in vitro with T7 RNA polymerase, using a PCR-amplified DNA template and HiScribe T7 Quick High Yield RNA Synthesis kit .
Residues 34–67 of ωRNA scaffold were replaced by a GAAA linker. The GAAA linker-fused boxB RNA was appended to the 3′ end of the ωRNA scaffold. The same target DNA strand was used for this reconstitution. The 5′ extended non-target DNA strand was purchased from GENEWIZ. The purified DtIsrB protein was mixed with the ωRNA, the target DNA strand, and the non-target DNA strand in buffer G and incubated at 37 °C for 15 min.
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