Targeting the weakest links in DNA for selective structural recognition

Deoxyribonucleic acid (DNA) is unique for each individual, it is linked to genetic disease, and normally forms a double helix. DNA is made up of four bases - adenine (A), thymine (T), guanine (G) and cytosine (C), and with A pairing with T and G pairing with C to give the familiar long chain with a right-handed twist. Nucleic acids (DNA and RNA) can actually adopt a number of different structures, which are greatly dependent on the order of the four bases. The sequence of the bases has a profound effect on the shape and stability of the DNA chain, with specific base steps (e.g. thymine followed by adenine) more flexible than others. Ruthenium polypyridyl complexes are small molecules that have been shown to insert themselves between the bases of DNA and have been investigated for a variety of applications, from light activated anti-cancer therapy to the detection of base mismatches (in which the bases are paired with the incorrect partner and which are linked to the onset of several diseases). These ruthenium complexes are prepared as mixtures of two different molecules (known as enantiomers), that only differ by the direction of the components surrounding the ruthenium centre, like propellers which can turn either way, to the right (clockwise) or to the left (anticlockwise). Much of the research performed on the ruthenium complexes has used an equal mixture of the enantiomers (due to the difficulty in separating the two molecules from one another; they are chemically identical except for this property). Our recent research has shown that the interaction of ruthenium complexes to DNA is not straightforward, with each enantiomer capable of binding to the DNA in a different manner, which could limit their use in the applications mentioned above. We propose to combine our experience in X-ray crystallography (producing solid crystals of DNA and complex that can be used to understand the arrangement of the two components relative to each other) with a systematic study of binding to specific sequences of DNA. We will use enantiomerically pure ruthenium complexes (in which one of the enantiomers is completely separated from the other) to understand how each molecule binds, and the relationship between the two. Experiments conducted in our laboratory, involving crystallography and techniques in solution, have shown that both enantiomers seem to target and bind weakness in the DNA structure and sequence, but sometimes in different ways. This weakness can be in the form of DNA that has been damaged either by the presence of mismatches (where the bases pair with the wrong partner), breaks in the DNA strand, or the presence of bases damaged by environmental conditions (for example chemicals or radiation). Another interesting property of nucleic acids is that they can adopt different structures, distinct from the well known right-twisting double helix. The ability to specifically recognise these alternative structures of nucleic acids will allow us to understand their role within the body, and to develop ways of using them to combat disease. Two of these more specialised structures are the G-quadruplex and i-motif structure, which are four stranded assemblies of DNA that can be formed by specific sequences found in our genetic code. There is a growing body of evidence that suggests that these structures may play important roles in several processes in the body, and being able to target them and selectively stabilise or destabilise could lead to new treatment for diseases as diverse as diabetes to cancer. The proposed research is important in order to unlock the potential of the ruthenium complexes as anti-cancer drugs, or selective biological probes. We must carry out the proposed research to make sense of the way they bind and the effect they then have on the DNA structure and its behaviour in the test tube.

Weakly bonded steps in DNA structure are known to be important for several biological processes involving molecular recognition. Weakness, due to weak stacking or hydrogen bonding, for example, can be associated with sequence dependent effects, mismatched base pairs, single strand breaks or base damage. We have identified that several ruthenium polypyridyl complexes can bind specifically to weak links in a DNA sequence, and aim to make use of this recognition and to investigate whether other forms of weakness can also be targeted. We will use a combination of X-ray crystallography and solution techniques including UV, CD and fluorescence spectroscopy to conduct a systematic study of ruthenium complex binding to a number of DNA systems, including standard Watson-Crick double helix, mismatched bases, single strand breaks, damaged bases, higher order DNA structures, as well as RNA structures. Using the atomic level structural information from crystallography and studying the same and related systems in solution is the best way for us to interpret the selective binding and any structural changes that occur. The Cardin group have successfully crystallised a range of DNA/ligand assemblies in the last twenty years or so, and used the resulting models to interpret data from a range of biophysical measurements on closely related systems, even though the process of crystallisation often yields surprises, and the structures give unexpected insights. The use of the Research Complex at Harwell combined with the recent availability of new screening kits for nucleic acid crystallisation have greatly helped the success rate and rational design of crystallisation experiments by the current team. The powerful combination in this approach will now allow us to rationally design modified metal complexes to maximise selectivity and properties such as fluorescence, used in sensing applications.

The fundamental nature of the proposed research has clear benefits to the academic community, with insights into the binding of enantiomers of ruthenium complexes to various nucleic acid structures. Behind this academic impact is a range of scientific techniques that require a high level of skill from the researchers involved, in order to fully exploit the limits of the techniques and ensure the correct understanding and interpretation of the results. The scientists involved in this research will enhance their skills within a variety of techniques, and be able to use these skills in subsequent employment. Several members of the Cardin group who have developed key skills in crystallography have gone on to obtain positions at world leading central facilities such as Diamond Light Source, and contribute to further research of national and international importance.
The detection and specific targeting of DNA damage, whether base mismatches, single strand breaks or damaged bases will have a range of uses in therapeutics and diagnostics. Ruthenium complexes have been considered promising candidates for photodynamic therapy for several years. The proposed research will enhance our understanding of the complex nature behind DNA binding, especially of the different enantiomers. If pharmaceutical companies are to develop these metal complexes into drugs for treatment of disease, then a full understanding of selective or specific binding is essential, along with knowledge of how derivatisation can tune the binding properties. The ability to selectively target and stabilise a specific motif in DNA, for example the weak 5'-TA-3' step, will allow us to probe the role of this motif in a number of biological process, for example the bending of DNA at the TATA box by proteins involved in transcription, which may lead to new therapeutic insights.
The awareness of the general public about DNA structure is often limited to that found in biology textbooks, most commonly the double helical nature of DNA. Some may be aware of the damage that chemicals can do to human health, but the underlying mechanisms and structural changes to DNA that occur are not as well known. The team in Reading have an expertise in understanding and visualising the structure of DNA and putting these structures into a biological context. Using our expertise to show how DNA can adopt multiple structures, and that these structure can be damaged to different degrees and in different ways, will help to educate the public to the importance of understanding how damage can occur and why it is important to protect our DNA from this damage, for example by wearing sun tan lotion, or refraining from smoking.