Patel D J, Canuel L L
Proc Natl Acad Sci U S A. 1976 Oct;73(10):3343-7. doi: 10.1073/pnas.73.10.3343.
The binding of ethidium bromide (EtdBr) to the dC-dG-dC-dG self-complementary duplex has been monitored at the resolvable drug and nucleic acid protons and backbone phosphates at high nucleotide/drug (N/D) ratios by nuclear magnetic resonance (NMR) spectroscopy in aqueous solution. We observe averaged resonances (25 degrees-95 degrees) for the nucleic acid and drug nonexchangeable protons in the presence of excess tetranucleotide (N/D = 24), indicative of rapid exchange relative to the chemical shifts in the free and complexed states. Complex formation results in upfield shifts for the base protons at the terminal and internal base pairs and an increase in the transition midpoint for the duplex-to-strand conversion. We observe upfield chemical shift changes of 1.2 ppm at the Watson-Crick guanine N-1 proton(s) on complex formation (N/D = 24), with slow exchange between (dC-dG-dC-dG)2 and EtdBr-(dC-dG-dC-dG)2 relative to this chemical shift difference at-5 degrees. The EtdBr phenanthridine ring protons shift upfield by about 0.9 ppm (H-2, H-4, H-7, H-9) and greater than 0.5 ppm (H-1, H-10) on complex formation, with the chemical shifts versus temperature plots (25 degrees-95 degrees) monitoring the dissociation of the EtdBr-(dC-dG-dC-dG)2 structure. These upfield shifts at the exchangeable and nonexchangeable base protons and phenanthridine ring (but not side chain) protons demonstrate intercalation of the phenanthridine ring of EtdBr into the dC-dG-dC-dG duplex in solution. The intercalation model may be supported by the observation of downfield shifts (up to 1ppm) at the internucleotide phosphate(s) of the tetranucleotide duplex on addition of EtdBr at low temperatures. We observe stronger binding of EtdBr to the self-complementary dC-dG-dC-dG (2 dC-dG intercalation sites) and dC-dC-dG-dG (1 dC-dG site) duplexes compared to the dG-dG-dC-dC (no dC-dG sites) as monitored by UV absorbance changes at 480 nm. These studies extend to the tetranucleotide duplex level earlier observations that EtdBr exhibits a selectivity for formation of complexes to dinucleoside monophosphates with a pyrimidine (3'-5') purine sequence in the crystal and in solution. The experimental proton NMR upfield shifts at the phenanthridine protons on formation of the EtdBr-(dC-dG-dC-dG)2 complex compare favorably with calculated values (atomic diamagnetic anisotropy and ring current contributions) based on the overlap geometry for EtdBr intercalated into the pyrimidine (3'-5') purine dinucleoside monophosphate duplex in the crystal.
在水溶液中，通过核磁共振（NMR）光谱法，在高核苷酸/药物（N/D）比率下，于可分辨的药物和核酸质子以及主链磷酸盐处监测了溴化乙锭（EtdBr）与dC-dG-dC-dG自互补双链体的结合情况。在存在过量四核苷酸（N/D = 24）的情况下，我们观察到了核酸和药物非交换质子的平均共振（25℃ - 95℃），这表明相对于游离态和复合态中的化学位移而言，存在快速交换。复合物的形成导致末端和内部碱基对处的碱基质子发生向高场位移，并且双链体向单链转化的转变中点增加。我们观察到在形成复合物（N/D = 24）时，Watson-Crick鸟嘌呤N-1质子处有1.2 ppm的向高场化学位移变化，相对于 - 5℃时的这种化学位移差异，(dC-dG-dC-dG)2与EtdBr-(dC-dG-dC-dG)2之间的交换缓慢。在形成复合物时，EtdBr菲啶环质子向高场位移约0.9 ppm（H-2、H-4、H-7、H-9）和大于0.5 ppm（H-1、H-10），通过化学位移与温度的关系图（25℃ - 95℃）监测EtdBr-(dC-dG-dC-dG)2结构的解离。可交换和不可交换碱基质子以及菲啶环（而非侧链）质子处的这些向高场位移表明，EtdBr的菲啶环在溶液中插入到dC-dG-dC-dG双链体中。在低温下添加EtdBr时，四核苷酸双链体的核苷酸间磷酸盐处出现向低场位移（高达1 ppm），这一观察结果可能支持插入模型。通过480 nm处的紫外吸收变化监测发现，与dG-dG-dC-dC（无dC-dG位点）相比，EtdBr与自互补的dC-dG-dC-dG（2个dC-dG插入位点）和dC-dC-dG-dG（1个dC-dG位点）双链体的结合更强。这些研究将早期的观察结果扩展到了四核苷酸双链体水平，即在晶体和溶液中，EtdBr对形成与具有嘧啶（3'-5'）嘌呤序列的二核苷单磷酸的复合物表现出选择性。在形成EtdBr-(dC-dG-dC-dG)2复合物时，菲啶质子处的实验性质子NMR向高场位移与基于晶体中插入到嘧啶（3'-5'）嘌呤二核苷单磷酸双链体中的EtdBr重叠几何结构计算得出的值（原子抗磁各向异性和环电流贡献）吻合良好。
