Tanious F A, Veal J M, Buczak H, Ratmeyer L S, Wilson W D
Department of Chemistry, Georgia State University, Atlanta 30303.
Biochemistry. 1992 Mar 31;31(12):3103-12. doi: 10.1021/bi00127a010.
The interaction of DAPI and propidium with RNA (polyA.polyU) and corresponding DNA (polydA.polydT) sequences has been compared by spectroscopic, kinetic, viscometric, Tm, and molecular modeling methods. Spectral changes of propidium are similar on binding to the AT and AU sequences but are significantly different for binding of DAPI. Spectral changes for DAPI with the DNA sequence are consistent with the expected groove-binding mode. All spectral changes for complexes of propidium with RNA and DNA and for DAPI with RNA, however, are consistent with an intercalation binding mode. When complexed with RNA, for example, DAPI aromatic protons signals shift significantly upfield, and the DAPI UV-visible spectrum shows significantly larger changes than when complexed with DNA. Slopes of log kd (dissociation rate constants) versus-log [Na+] plots are similar for complexes of propidium with RNA and DNA and for the DAPI-RNA complex and are in the range expected for an intercalation complex. The slope for the DAPI-DNA complex, however, is much larger and is in the range expected for a groove-binding complex. Association kinetics results also support an intercalation binding mode for the DAPI-RNA complex. The viscosity of polyA.polyU solutions increases significantly on addition of both propidium and DAPI, again in agreement with an intercalation binding mode for both molecules with RNA. Molecular modeling studies completely support the experimental findings and indicate that DAPI forms a very favorable intercalation complex with RNA. DAPI also forms a very stable complex in the minor groove of AT sequences of DNA, but the stabilizing interactions are considerably reduced in the wide, shallow minor groove of RNA. Modeling studies,thus,indicate that DAPI interaction energetics are more favorable for minor-groove binding in AT sequences but are more favorable for interaction in RNA.
通过光谱、动力学、粘度测定、熔点以及分子建模方法，比较了4',6-二脒基-2-苯基吲哚（DAPI）和碘化丙啶与RNA（聚腺苷酸·聚尿苷酸）及相应DNA（聚脱氧腺苷酸·聚脱氧胸苷酸）序列的相互作用。碘化丙啶与AT和AU序列结合时的光谱变化相似，但与DAPI结合时则有显著差异。DAPI与DNA序列结合时的光谱变化与预期的沟槽结合模式一致。然而，碘化丙啶与RNA和DNA形成的复合物以及DAPI与RNA形成的复合物的所有光谱变化均与插入结合模式一致。例如，当与RNA复合时，DAPI的芳族质子信号显著向高场移动，并且DAPI的紫外可见光谱显示出比与DNA复合时更大的变化。碘化丙啶与RNA和DNA形成的复合物以及DAPI-RNA复合物的log kd（解离速率常数）对-log [Na+]图的斜率相似，且处于插入复合物预期的范围内。然而，DAPI-DNA复合物的斜率要大得多，处于沟槽结合复合物预期的范围内。缔合动力学结果也支持DAPI-RNA复合物的插入结合模式。添加碘化丙啶和DAPI后，聚腺苷酸·聚尿苷酸溶液的粘度均显著增加，这同样与这两种分子与RNA的插入结合模式一致。分子建模研究完全支持实验结果，并表明DAPI与RNA形成了非常有利的插入复合物。DAPI在DNA的AT序列小沟中也形成了非常稳定的复合物，但在RNA宽而浅的小沟中，稳定相互作用大大降低。因此，建模研究表明，DAPI的相互作用能量学在AT序列中更有利于小沟结合，但在RNA中更有利于相互作用。
