James Peter L, Le Strat Loic, Ellervik Ulf, Bratwall Christina, Nordén Bengt, Brown Tom, Fox Keith R
Division of Biochemistry and Molecular Biology, School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton SO16 7PX, UK.
Biophys Chem. 2004 Nov 1;111(3):205-12. doi: 10.1016/j.bpc.2004.06.001.
We have used DNase I footprinting and fluorescence melting studies to study the interaction of the hairpin polyamide Im-Py-Py-Py-(R)H2Ngamma-Im-Py-Py-Py-beta-Dp with its preferred binding sites (5'-WGWWCW; W=A or T) and other sequences. DNase I footprinting confirmed that the ligand binds to the sequence AGAACA at nanomolar concentrations and that changing the terminal A to G causes a dramatic decrease in affinity, while there was no interaction with the reverse sequence WCWWGW. Fluorescence melting studies with 11-mer duplexes showed that the polyamide had very different effects on the forward (TGWWCT) and reverse (TCTAGT) sequences. At low concentrations, the polyamide produced biphasic melting curves with TGATCT, TGTACT and TGAACT, suggesting a strong interaction. In contrast, the melting profiles with TCTAGT were always monophasic and showed much smaller concentration dependent changes in Tm. The polyamide also showed weak binding to the sequence TGATCT when one of the central AT pairs was replaced with an AC mismatch. These melting profiles were compared with those produced by the AT-selective minor groove binding agents distamycin and Hoechst 33258 at the same sites and at similar sequences containing A5 and (AT)3, which are expected to bind distamycin in the 1:1 and 2:1 modes, respectively. These ligands produced simple monophasic melting curves in which the Tm steadily increased as the ligand concentration was raised.
我们已使用DNA酶I足迹法和荧光熔解研究来探究发夹型聚酰胺Im-Py-Py-Py-(R)H2Nγ-Im-Py-Py-Py-β-Dp与其偏好结合位点（5'-WGWWCW；W = A或T）及其他序列之间的相互作用。DNA酶I足迹法证实，该配体在纳摩尔浓度下与AGAACA序列结合，将末端的A替换为G会导致亲和力显著下降，而与反向序列WCWWGW没有相互作用。对11聚体双链体进行的荧光熔解研究表明，聚酰胺对正向序列（TGWWCT）和反向序列（TCTAGT）有非常不同的影响。在低浓度下，聚酰胺与TGATCT、TGTACT和TGAACT产生双相熔解曲线，表明存在强相互作用。相比之下，TCTAGT的熔解曲线始终为单相，且在熔点（Tm）上显示出小得多的浓度依赖性变化。当中心的一对AT被AC错配取代时，聚酰胺与TGATCT序列也表现出弱结合。将这些熔解曲线与AT选择性小沟结合剂偏端霉素和Hoechst 33258在相同位点以及含有A5和(AT)3的类似序列处产生的熔解曲线进行比较，预计它们分别以1:1和2:1的模式与偏端霉素结合。这些配体产生简单的单相熔解曲线，其中随着配体浓度升高，Tm稳步增加。
