Binding of distamycin A and netropsin to the 12mer DNA duplexes containingmixed AT center dot GC sequences with at most five or three successive AT base pairs

Circular dichroism (CD), isothermal calorimetric titrations (ITC), and temp erature-dependent UV spectroscopy were used to investigate binding of the m inor groove-directed ligands distamycin A (Dst) and netropsin (Net) to the following duplexes: d(GTTAGTATTTGG) d(CCAAATACTAAC),.d(GTTAGTATATGG) d(CCAT ATACTAAC),.d(GTTAGTACTTGG) d(CCAAGTACTAAC), and d(GTTAGTAGTTGG).d(CCAACTACT AAC). Our results reveal that Dst binds within the minor grooves of these d odecamers that contain five-AT and/or four-AT GC binding sites exclusively in a dimeric high-affinity 2:1 binding mode (K approximate to 10(16) M-2). By contrast, Net exhibits high-affinity binding only when it binds in a 1:1 mode (K-1 approximate to 10(9) M-1) to the two duplexes that contain five- AT sites (5'-TATTT-3' and 5'-TATAT-3'). Its further binding to these two du plexes occurs in a low-affinity mode (K-2 approximate to 10(6) M-1) and res ults in the formation of 2:1 Net-DNA complexes. To the other two duplexes t hat contain sequences with at most three AT consecutive base pairs Net bind s in two distinctive low-affinity 1:1 binding modes (K-1 approximate to 10( 7) M-1, K-2 approximate to 10(6) M-1). Competition experiments (CD and ITC titrations) reveal that Dst entirely displaces Net from its 1:1 and 2:1 com plexes with any of the four duplexes. We discuss and interpret our optical and calorimetric results in the context of the available structural informa tion about the complexes between DNA and the sequence-specific minor groove binders Dst and Net.