Metal polypyridyl complex interactions with duplex and higher order DNAs

双螺旋结构完全可以被称为科学的标志。从洗发水到高科技医疗设备，这些缠绕在一起的头发的图片被用于销售各种产品。然而，对于与DNA结合的分子如何在分子水平上改变其结构，我们所知甚少。许多是通过插入来实现的，也就是说，通过在DNA碱基对堆栈中增加一个额外的步骤，在碱基对之间滑动。这种结合模式延长了DNA链，可能导致DNA损伤或突变，但在癌症治疗中也有治疗益处。插入也可以构成检测DNA方法的基础。一些分子，其中最著名的是乙二胺，嵌入并发出荧光，这使得它们很容易被观察到，DNA本身是无色的，在电磁波谱的可见区域内不会发出荧光。我们最近研究的钌“光开关”配合物显示出比乙啶更强的荧光，但是，作为金属配合物和手性（显示手性），它们是一个更复杂的系统，难以理解。我们最近已经证明，这些复合物是从DNA的小凹槽插入的。我们还表明，确切的结合模式取决于DNA步骤，这在设计特定DNA序列的探针时很重要。例如，如果左旋分子在胸腺嘧啶-腺嘌呤步骤结合，它嵌入得很深，无法形成任何其他相互作用。然而，如果它在鸟嘌呤-鸟嘌呤步骤结合，因为鸟嘌呤碱基更大，钌络合物不能如此深入地插入小凹槽。这意味着复合体的第二个“臂”可以与第二个DNA双链相互作用，在这一步引起扭结，这被称为半嵌入。这种扭结行为可能发生在其他分子上，但这是第一次直接可视化。我们还不确定这些特征是否可以直接用于癌症和相关治疗，但更直接的应用类型是对特定DNA序列和形状的非常敏感的检测。在目前的提案中，我们的目标是，最重要的是，第一次定义，并利用我们目前在该领域的世界领先地位，钌配合物在双螺旋结构的特定步骤中“到位”所必需的精确形状拟合。为了理解这一点，我们将研究具有可变形状配体的钌配合物，结合双工形成的DNA序列，以及一些高阶结构，如四重结构（g -四重体）和i基序，这些结构在端粒染色体末端的单链DNA中发现。这些特殊的结构与基因开关有关，据估计，人类基因组中可能有40万个这样的区域可以形成这样的结构。我们将进行互补溶液研究，因为一个经常出现的问题是，我们如何知道一个分子在晶体的“x射线透镜”中是否会在试管中（或者实际上，在活细胞中）表现出相同的行为。一些溶液测量对一些因素很敏感，比如分子在结合袋中的方向、接触水的量、另一个分子是否在邻近的位置结合、DNA序列、pH值、存在的盐等等。因此，将晶体的致密和不动的世界与光谱学家使用的快速变化的稀释溶液的世界联系起来是很重要的，两者中哪一个更接近染色体的紧密超卷曲和缠绕的DNA，这也许是一个有争议的问题。只有通过研究晶体，我们才能知道任何分子的细节，这些细节必须与这些不同的观点分享和联系起来。

The double helix could be justifiably be called a scientific icon. Graphics showing the intertwined strands are used to market everything from hair shampoo to high-tech medical equipment. Yet we know remarkably little about exactly how molecules which are known to bind to DNA change its structure at the molecular level. Many do so by intercalation, that is, by adding an extra step to the DNA base stack, slipping in between the base pairs. This mode of binding lengthens the stack, and may cause DNA damage or mutation, but can also be of therapeutic benefit in cancer treatment.Intercalation can also form the basis of a method for detecting DNA. Some molecules, of which the best known is ethidium, intercalate and fluoresce, which makes them easy to visualise, DNA itself being colourless and not fluorescing in the visible region of the electromagnetic spectrum. The ruthenium 'light-switch' complexes we have recently studied show a much stronger fluorescence than ethidium, but, being metal complexes and chiral (showing handedness) they are a more complex system to understand. We have very recently shown that these complexes intercalate from the minor groove of DNA. We have also shown that the exact mode of binding depends on the DNA step, which is important in designing probes for specific DNA sequences. For example, if the left-handed molecule binds at a thymine-adenine step, it intercalates so deeply that it cannot form any other interactions. If, however, it binds at a guanine-guanine step, because the guanine base is larger, the ruthenium complex cannot intercalate so deeply into the minor groove. This means that a second 'arm' of the complex can interact with a second DNA duplex, causing kinking at that step, which is called semi-intercalation. This kinking behaviour probably occurs with other molecules, but this is the first time it has been directly visualised. We are not sure yet, whether these features can be put to direct use in cancer and related therapies, but a more immediate type of application is in the very sensitive detection of particular DNA sequences and shapes.In the present proposal, we aim, most importantly, to define for the first time, and making use of our currently world-leading position in the field, the exact shape-fitting which is necessary for the ruthenium complexes to 'click into place' at a particular step in the double helix. To understand this, we will look at ruthenium complexes having variable-shaped ligands, in combination with duplex-forming DNA sequences, together with some higher-order structures such as the quadruplexes (G-quartets) and the i-motif, found in single-stranded DNA at the ends of chromosomes in the telomeres. These specialised structures are associated with genetic switches, and it is estimated that there are perhaps 400,000 such regions in the human genome where such structures could form.We will carry out complementary solution studies, since a frequent question which arises is how we can know whether a molecule caught in the 'X-ray lens' of a crystal will show the same behaviour in the test tube (or indeed, in the living cell). Several solution measurements are sensitive to factors such as the orientation of a molecule in its binding pocket, the amount of access to water, whether or not another molecule is bound in an adjacent site, the DNA sequence, the pH, the salts present, and so forth. It is therefore important to relate the compact and immobile world of the crystal to that of the rapidly changing dilute solution used by spectroscopists, and it is a moot point, perhaps, which of the two may be closer to the tightly supercoiled and wound DNA of the chromosome.It is only by studying the crystal that we can know any molecular detail, which insight has then to be shared and related to these alternative viewpoints.