Bailly Christian, Arafa Reem K, Tanious Farial A, Laine William, Tardy Christèle, Lansiaux Amélie, Colson Pierre, Boykin David W, Wilson W David
INSERM U-524 et Laboratoire de Pharmacologie Antitumorale du Centre Oscar Lambret, IRCL, 59045 Lille, France.
Biochemistry. 2005 Feb 15;44(6):1941-52. doi: 10.1021/bi047983n.
The phenanthridinium dye ethidium bromide is a prototypical DNA intercalating agent. For decades, this anti-trypanosomal agent has been known to intercalate into nucleic acids, with little preference for particular sequences. Only polydA-polydT tracts are relatively refractory to ethidium intercalation. In an effort to tune the sequence selectivity of known DNA binding agents, we report here the synthesis and detailed characterization of the mode of binding to DNA of a novel ethidium derivative possessing two guanidinium groups at positions 3 and 8. This compound, DB950, binds to DNA much more tightly than ethidium and exhibits distinct DNA-dependent absorption and fluorescence properties. The study of the mode of binding to DNA by means of circular and electric linear dichroism revealed that, unlike ethidium, DB950 forms minor groove complexes with AT sequences. Accurate quantification of binding affinities by surface plasmon resonance using A(n)T(n) hairpin oligomer indicated that the interaction of DB950 is over 10-50 times stronger than that of ethidium and comparable to that of the known minor groove binder furamidine. DB950 interacts weakly with GC sites by intercalation. DNase I footprinting experiments performed with different DNA fragments established that DB950 presents a pronounced selectivity for AT-rich sites, identical with that of furamidine. The replacement of the amino groups of ethidium with guanidinium groups has resulted in a marked gain of both affinity and sequence selectivity. DB950 provides protection against DNase I cleavage at AT-containing sites which frequently correspond to regions of enhanced cleavage in the presence of ethidium. Although DB950 maintains a planar phenanthridinium chromophore, the compound no longer intercalates at AT sites. The guanidinium groups of DB950, just like the amidinium group of furamidine (DB75), are the critical determinants for recognition of AT binding sites in DNA. The chemical modulation of the ethidium exocyclic amines is a profitable option to tune the nucleic acid recognition properties of phenylphenanthridinium dyes.
菲啶鎓染料溴化乙锭是一种典型的DNA嵌入剂。几十年来，这种抗锥虫剂一直被认为能嵌入核酸，对特定序列几乎没有偏好。只有聚dA - 聚dT片段相对难以被溴化乙锭嵌入。为了调节已知DNA结合剂的序列选择性，我们在此报告一种新型溴化乙锭衍生物的合成及其与DNA结合模式的详细表征，该衍生物在3位和8位具有两个胍基。这种化合物DB950与DNA的结合比溴化乙锭紧密得多，并表现出独特的依赖于DNA的吸收和荧光特性。通过圆二色性和电线性二色性研究与DNA的结合模式表明，与溴化乙锭不同，DB950与AT序列形成小沟复合物。使用A(n)T(n)发夹寡聚物通过表面等离子体共振对结合亲和力进行准确量化表明，DB950的相互作用比溴化乙锭强10 - 50倍以上，与已知的小沟结合剂呋咱米定相当。DB950通过嵌入与GC位点弱相互作用。用不同DNA片段进行的DNase I足迹实验表明，DB950对富含AT的位点具有明显的选择性，与呋咱米定相同。用胍基取代溴化乙锭的氨基导致亲和力和序列选择性都有显著提高。DB950能保护含AT的位点免受DNase I切割，而在溴化乙锭存在时这些位点通常对应于切割增强的区域。尽管DB950保持平面菲啶鎓发色团，但该化合物不再在AT位点嵌入。DB950的胍基，就像呋咱米定（DB75）中的脒基一样，是识别DNA中AT结合位点的关键决定因素。对溴化乙锭环外胺进行化学调节是调节苯基菲啶鎓染料核酸识别特性的一个有益选择。
