McPike Mark P, Goodisman Jerry, Dabrowiak James C
Department of Chemistry, Center for Science and Technology, R 1-014,Syracuse University, Syracuse, NY 13224-4100, USA.
Bioorg Med Chem. 2002 Nov;10(11):3663-72. doi: 10.1016/s0968-0896(02)00220-1.
We have studied the interaction of the aminoglycoside drug, paromomycin, with a 171-mer from the packaging region of HIV-1 (psi-RNA), using quantitative footprinting and circular dichroism spectroscopy. The footprinting autoradiographic data were obtained by cutting end-labeled RNA with RNase I or RNase T1 in the presence of varying paromomycin concentrations. Scanning the autoradiograms produced footprinting plots showing cleavage intensities for specific sites on the psi-RNA as functions of drug concentration. Footprinting plots showing binding were analyzed using a two-state model to give apparent binding constants for specific sites of the psi-RNA. These plots show that the highest-affinity paromomycin binding site involves nucleotides near bulges in the main stem and SL-1, and other nucleotides in SL-4 of the psi-RNA. RNase I gives an apparent value of K for this drug site of approximately 1.7 x 10(5) M(-1) while RNase T1 reports a value of K of approximately 8 x 10(4) M(-1) (10 mM Tris HCl, pH 7). Footprinting shows that loading the highest affinity site with paromomycin causes structural changes in the single-stranded linker regions, between the stem-loops and main stem and the loops of SL-1 and SL-3. Drug-induced structural changes also affect the intensity of the 208 nm band in the circular dichroism spectrum of the psi-RNA. Fitting the changes in CD band intensity to a two-state model yielded a binding constant for the highest-affinity drug site of 6 x 10(6) M(-1). Thus, the binding constants from footprinting are lower than those obtained for the highest-affinity site from the circular dichroism spectrum, and lower than those earlier obtained using absorption spectroscopy (Sullivan, J. M.; Goodisman, J.; Dabrowiak, J. C., Bioorg. Med. Chem. Lett. 2002, 12, 615). The discrepancy may be due to competitive binding between drug and cleavage agent in the footprinting experiments, but other explanations are discussed. In addition to revealing sites of binding and regions of drug-induced structural change, footprinting showed that the loop regions of SL-1, SL-3 and SL-4 are exposed in the RNA, whereas the linker region between SL-1 and SL-2 is 'buried' and not accessible to cutting by RNase I or RNase T1.
我们使用定量足迹法和圆二色光谱法，研究了氨基糖苷类药物巴龙霉素与来自HIV-1包装区域的171聚体（ψ-RNA）之间的相互作用。足迹放射自显影数据是通过在不同浓度的巴龙霉素存在下，用核糖核酸酶I或核糖核酸酶T1切割末端标记的RNA获得的。扫描放射自显影片产生足迹图，显示ψ-RNA上特定位点的切割强度作为药物浓度的函数。使用双态模型分析显示结合的足迹图，以给出ψ-RNA特定位点的表观结合常数。这些图表明，巴龙霉素的最高亲和力结合位点涉及主干和SL-1中凸起附近的核苷酸，以及ψ-RNA的SL-4中的其他核苷酸。对于该药物位点，核糖核酸酶I给出的K表观值约为1.7×10⁵ M⁻¹，而核糖核酸酶T1报告的K值约为8×10⁴ M⁻¹（10 mM Tris HCl，pH 7）。足迹法表明，用巴龙霉素加载最高亲和力位点会导致茎环与主干之间以及SL-1和SL-3环的单链连接区域发生结构变化。药物诱导的结构变化也会影响ψ-RNA圆二色光谱中208 nm波段的强度。将CD波段强度的变化拟合到双态模型中，得到最高亲和力药物位点的结合常数为6×10⁶ M⁻¹。因此，足迹法得到的结合常数低于从圆二色光谱中获得的最高亲和力位点的结合常数，也低于早期使用吸收光谱法获得的结合常数（Sullivan，J. M.；Goodisman，J.；Dabrowiak，J. C.，Bioorg. Med. Chem. Lett. 2002，12，615）。这种差异可能是由于足迹实验中药物与切割剂之间的竞争性结合，但也讨论了其他解释。除了揭示结合位点和药物诱导的结构变化区域外，足迹法还表明，SL-1、SL-3和SL-4的环区域在RNA中是暴露的，而SL-1和SL-2之间的连接区域是“埋藏”的，核糖核酸酶I或核糖核酸酶T1无法切割。
