Figure 7.
Vc2* RNA binding to 32P-cdiGMP is detected by DRaCALA. (A) Spots visualized by phosphorimager with streptavidin used to immobilize biotinylated RNA. The binding reaction contained 4 nM 32P-cdiGMP, 1 µM RNA and 200 nM streptavidin in buffer (10 mM KCl, 10 mM sodium cacodylate, 3 mM MgCl2). (B) The affinity of Vc2*-biotin RNA for cdiGMP was determined with both EMSA and DRaCALA by diluting RNA in the binding reaction. The fraction bound is normalized such that 1.0 represents maximal binding. The DRaCALA-obtained affinity was Kd = 7.8 ± 1.9 × 10−9 M and the apparent affinity in EMSA was Kd = 9.8 ± 1.6 × 10−9 M.