Metal polypyridyl complex interactions with duplex and higher order DNAs

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.

Lambda-Ru(phen)2(dppz)]2+ (dppz=dipyridophenazine) has been shown (Cardin et al. Nature Chemistry, 2012, 4:621-628) to bind to the DNA duplex d(CCGGTACCGG)2. X-ray crystallography showed symmetrical intercalation from the minor groove at the central TA/TA step, and angled intercalation at the terminal CC/GG steps. The second of these modes was also seen in the (TCGGCGCCGA)2 duplex with lambda-Ru[(TAP)2(dppz)]2+ (TAP=tetraazaphenanthrene) (Cardin et al. PNAS 2011, 108:17571-17856). Both structures also showed kinking of the DNA at the GG/CC steps due to semi-intercalation by one of the TAP or phen ligands. We propose to
1. Define the sequence specificity of these three binding modes.
2. Extend our results to a range of mono and diruthenium complexes.
3. Use a range of spectroscopic techniques to correlate crystal and solution data.
4. Seek to understand the structural origins of the 'light-switch' effect with further structural studies of both the ground and the excited states of the bound complexes.
5. Exploit the potential of these metal cations for molecular recognition of higher-order DNA, e.g. the i-motif.

Who will benefit and how?
X-ray crystallography is by its nature a fundamental scientific technique, which does not of itself directly lead to impact. For example, the determination of the structure of the ribosome was a recent triumph, resulting in Nobel prizes for Ramakrishnan, Yonath and Steitz in 2009. The ribosome is the target for drugs such as aminoglycosides, macrolides, spectinamides and tetracyclines, and once the structure is known, it is then possible to carry out structure-based drug design on compounds of interest. The impact on society is only realised if a better drug can be developed, but there are many steps in such a process. We must consider the impact of our proposed research within this context.
The 'light switch' properties of ruthenium complexes apply themselves to areas of sensing, signalling, diagnostics and therapeutics, with photodynamic therapy one such example (the treatment of tumours using photoactivatable compounds). A fuller understanding of the sequence selectivity and how this will affect the signalling properties of the metal complexes will allow greater exploitation of this technology within medical applications such as DNA sensing. The effect of sequence binding to the fluorescent properties of the ruthenium complex will also deliver increased information for use in photodynamic therapy. Pharmaceutical companies will begin to have a greater understanding of how these complexes can be tailored to recognise specific sequences and particular concentations of nucleic acid within cells (e.g. mitochondrial or nuclear DNA) and therefore increase the likelihood of their development into clinically useful products. The extension of the research, into targeting other DNA structures, such as DNA quadruplexes, will further drive this work to the attention of pharmaceutical companies, with intense interest in G-quadruplex binding ligands currently evident.
The Cardin group are world leaders in the crystallisation of DNA/ruthenium complex systems, a position that is unlikely to be challenged in the short term. This is mainly because there are only a few groups worldwide specialising in this sort of nucleic acid crystallography, which is on a tiny molecular scale compared to the ribosome, but which can yield a high level of detail. In the last 18 months or so, the Cardin group has published the first two high profile papers, showing how this class of metal complexes bind to duplex DNA (Cardin et al.(2011) Proc. Natl. Acad. Sci. U.S.A., 108, 17610-17614. Cardin et al. (2012) Nature Chem,. 4, 621-628). In both cases the papers were cover stories (designed by CJC and JPH). In both cases, the Editor commissioned a Commentary article to run alongside our work, and in the case of Nature Chemistry, our paper was the subject of an Editorial and a commissioned interview as well. We therefore feel these papers can justifiably be described as 'high impact', and the first (published October 25th 2011) has already received 12 citations. The PNAS paper was highlighted in the Diamond Annual Review 2011-12, and the Nature Chemistry paper the cover story of the current Autumn 2012 Diamond News. It is clear from this recent activity that this team has the ability to generate world class research communication and would be capable of attracting R & D resources into the UK as the therapeutic interest in this research increases as described above. The skills developed by the researchers involved in this project will also place them in a very strong position to gain employment in any number of research organisations, greatly contributing to the skilled workforce within the UK.