Metal polypyridyl complex interactions with duplex and higher order DNAs

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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.