Bacterial rRNA A-site recognition by DAPI: Signatures of intercalative binding

The bacterial A-site RNA is one of the key targets towards the development of new antibacterials including new treatment options for tuberculosis. Using DAPI as a prototype, we have explored the potential of bisamidines as a potential chemical motif for bacterial A-site recognition. We have demonstrated that the binding of DAPI shows a concentration-dependent thermal stabilization of the bacterial A-site RNA (ΔT_m = 9.9 °C). The binding, however, does not show pH-dependent changes upon lowering of pH. Both UV-vis and CD experiments show that the DAPI binding involves base stacking with the RNA bases in a manner that is analogous to intercalation. Scatchard analysis of the UV-vis titration data revealed a micromolar affinity of the DAPI to the bacterial rRNA A-Site (K_a = 1.14 × 10⁶ M^-1) which was corroborated by the FID-based relative binding affinity comparison with aminoglycosides. The molecular docking studies showed binding poses consistent with polar and stacking interactions with the RNA. These studies highlight the role of amidines in bacterial A-site recognition and the need for the development of their structural analogs towards the making of aminoglycoside mimics.