Non-covalent interaction between CA-TAT and calf thymus DNA: Deciphering the binding mode by in vitro studies

CA-TAT, a novel peptide analog, was modified at the N-terminus of TAT (47-57), the cell-penetrating peptide transacting activator of transcription, by attaching cecropin A (1-7). CA-TAT, TAT (47-57), and cecropin A (1-7) were synthesized using standard Fmoc solid-phase peptide synthesis procedures, purified using reversed-phase high performance liquid chromatography (RP-HPLC), and characterized using ESI-MS. CA-TAT demonstrated antibacterial activities against bacteria with low hemolysis (MHC > 128 μM). The minimum inhibitory concentration (MIC) values of CA-TAT were in the range of 1-16 μM, which completely inhibited both gram-positive and gram-negative bacteria. The interactions between CA-TAT or TAT (47-57) and calf thymus DNA (ct-DNA) were investigated using multi-spectroscopic techniques and viscometry. The results showed that both CA-TAT and TAT (47-57) can interact with DNA via the minor groove-binding mode, and binding constant was calculated to be 2.83 × 10⁵ L mol^-1 at 310 K, which is lower than that with the classical intercalation binder ethidium bromide (EB). Compared with TAT (47-57) or cecropin A (1-7), CA-TAT combined with DNA much closer. The study results suggest that CA-TAT can be used as a novel antibacterial peptide in the development of new antibiotics because of its antibacterial activity that targets intracellular DNA.