Typhoon scanner system

In vitro-transcribed RNAs of pcry5Ba (200 nt, −100 to +100 regions of cry5Ba mRNA including the ribosome binding site, RBS) and pcry5BaM (187 nt, −100 to +100 regions of cry5Ba mRNA without the predicted 13 base pairs that interact with BtsR1) were created. Full-length BtsR1 RNA was synthesized with a FAM label at the 5′ end (AuGCT, Beijing). RNAs were assessed with an RNA6000 NANO chip on the Agilent Bioanalyzer 2100 system. FAM-BtsR1 (10 nM) was incubated in the presence or absence of pcry5Ba RNA (0, 100, 500, 1000, 1500, 2000, 2500 and 5000 nM) or pcry5BaM RNA (0, 100, 500, 1000, 1500, 2000, 2500 and 5000 nM) for the EMSA reactions, which were performed as previously described (29 (link)). Samples were electrophoresed through a 5% Tris/Borate/EDTA (TBE) gel at 200 V for 25 min, and then transferred to a positively charged nylon membrane and UV-crosslinked. The membrane was blocked and washed, and the labeled RNA was detected with a Typhoon Scanner system (GE, Healthcare).

The RNAs of pcry5Ba and pcry5BaM as well as seven selected ΔBtsR1 mutants were transcribed in vitro. FAM–BtsR1 (10 nM) was incubated in the presence or absence of pcry5Ba RNA (0 to 1000 nM) to conduct the competitive EMSA reactions. Then, the seven selected, unlabeled BtsR1 mutants were added into each reaction system at a concentration of 5000 nM to compete with the FAM−BtsR1 interaction with pcry5Ba RNA. Reactions were performed as previously described (Liu et al., 2012). Samples were electrophoresed through a 5% TBE mini-gel at 200 V for 25 min, then transferred to a positively-charged nylon membrane and UV-crosslinked. The membrane was blocked and washed, and the labeled RNA was detected using a Typhoon Scanner system (GE Healthcare).