ETNA - Expansion of the Time domain in Nucleic Acid crystallography

Lead Research Organisation: Diamond Light Source
Department Name: Science Division


The iconic double helix structure of DNA was confirmed in a single crystal structure in 1980, once it was possible to make small synthetic segments of DNA to order, and to purify them to a state where they could be crystallised. At that stage relatively large crystals, maybe 0.5 mm in size, would be necessary, because the power of X-ray sources was still rather limited. Current technology, such as that available at Diamond Light Source, means that we can see individual carbon atoms in such small sections of DNA with a precision of picometres, even in a crystal at room temperature and bathed in an aqueous solution. We can do this over a range of temperatures, including room temperature, with varying degrees of hydration, and on crystals as small as 5 micron size. By using shorter wavelengths and larger crystals, 0.1 mm or so, we can also measure very accurate atomic positions, because a short wavelength in effect extends the detail we can see (increased resolution), even perhaps extending to the direct observation of hydrogen atoms.
Over the same sort of timescale, ultrafast laser measurements have developed to the point where they can be used to track the movement of electrons after excitation by light of a molecule, on a picosecond timescale, and extremely tiny differences in absorption of light can be monitored. These tiny differences relate to the differences in electron distribution in excited states of molecules. Recently this technique has been used, for example, to study the earliest steps in DNA damage caused by photosensitisers, as well as direct damage, similar to that caused by exposure to the sun's harmful UVA and UVB rays. One such system is that formed by the ruthenium polypyridyl 'light-switch' and related complexes. We have recently published the first crystal structures of these compounds bound to a DNA duplex, and are therefore in a uniquely strong position to study these systems.
We now propose to combine these two powerful techniques to track for the first time, not just fast DNA excitation and possible damage by light, but also to pinpoint exactly where this excitation occurs. We will make use of our expertise in preparing suitable crystals to do this for both photosensitised and direct DNA damage. In an extension of this initial idea (for which we already have enough preliminary data to be confident that our method of sample preparation gives useful results), we will use selective chemical modification of some of these systems. The DNA bases have been described as 'nature's sunscreens' because they appear to have been selected for their resistance to damage by direct irradiation, but small modifications (such as replacing the guanine 6-carbonyl group with the thiocarbonyl sulfur analogue) result in greatly lengthened photochemical lifetimes, with correspondingly greater potential for DNA damage, and this is indeed a known side-effect of some compounds used as sensitisers in photodynamic therapy. The new ultrafast technology of the LIFEtime instrument, currently under construction, will be used by us to monitor the successive steps in some of these damage processes. Ideally we would like to be able to track the formation of well known damage products such as 8-oxoguanine, and for this experiment we will use photosensitisers which are known to cause damage by this mechanism. One such metal-containing photosensitiser is a rhenium complex, having some chemical features in common with the ruthenium complexes.

The combination of crystallography and ultrafast kinetic measurements should give us a 'movie' of the process of light-induced DNA damage.

Technical Summary

We recently carried out several X-ray crystallographic studies of the binding of ruthenium polypyridyl complexes to DNA duplexes, as well as the first preliminary experiments to relate the X-ray data to ultrafast kinetic data on the photoexcitation of the bound complexes (with JMK and SQ). Our most recent crystallographic study (Cardin et al, J. Am. Chem. Soc. 2013, 135, 12652-12659), of both enantiomers of the 'light-switch' complex [Ru(phen)2(dppz)]2+ (dppz= dipyridophenazine) bound to the DNA hexamer duplex d(ATGCAT)2 showed for the first time the binding of any enantiomeric pair to DNA.
Combining these approaches, and building on our recently granted ULTRA facility access (CRuX programme) as well as the future LIFEtime instrument, we now propose to carry out ultrafast (TA and ps-TRIR) measurements on a range of well characterised and reproducibly crystallisable nucleic acid systems (with JMK and SQ). These measurements will be complemented by VCD data.
Among systems we plan to study are
1. The d(TCGGCGCCGA)2/lambda-[Ru(TAP)2(dppz)]2+ system (TAP=tetraazaphenanthrene), already the subject of the PNAS cover story (October 25th 2011) for its description of DNA kinking by semi-intercalation of the TAP ligand. Our preliminary studies reveal that transients corresponding to the formation of the guanine radical cation can indeed be observed.
2. A range of crystals with similar crystal packing but with local variations in DNA sequence and in metal complex, thus showing the kinetic pattern.
3. In the longer term, the LIFEtime system (which will enable a reaction course to be tracked) will be used to follow a specific DNA damage process, such as the formation of 8-oxoguanine in the presence of specific DNA sequences and appropriate metal complexes
4. The possibilities for direct photoexcitation of specific DNA structural motifs.

Planned Impact

The project brings together the world leading crystallographic facilities of Diamond Light Source with those of the Lasers for Science facility. It will provide an excellent intellectual forum for a meeting of minds between the facilities, and a superb career opportunity for JPH.

The skills contributing to this project include nucleic acid crystallization, synchrotron X-ray crystallography, synthetic coordination chemistry, nucleic acid chemistry, ultrafast laser physics, photochemical kinetics and circular dichroism, including the rare vibrational circular dischroism, a powerful array of techniques which together will allow us to look in unrivalled detail at one set of processes which up till now have chiefly been studied in dilute solution and with DNA from a range of sources, many of them unsuited to an unambiguous interpretation of the resulting spectroscopic information. For this information to be useful in the design of drugs for photodynamic therapy or for DNA sensing within living cells, it is necessary to have a clear description of the exact binding of the compound, together, ideally, with some insight as to how the binding relates to the reactivity.
What we aim to do is to show how a detailed knowledge of structure can be applied to interpret spectroscopic data, by carrying out the spectroscopic measurements directly on the crystals. Up till now, the interpretation of data from techniques such as ps-TRIR has been challenging because of the multiple conformations of DNA in solution and lack of knowledge of how exactly the ligands might be bound. It is in this area where the world leading team of JMK and SQ will have a major impact, by guiding us to an interpretation of the data.

Such fundamental knowledge can be applied to gain specific insights in specific cases e.g. 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 of the excitation, and the subsequent decay processes, 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.

We expect that the publication of our first results, within the first year of the proposed timescale, will provide an opportunity for impact. We would expect these results to be accompanied by a University press release, together with a place in the Diamond Annual Report and the corresponding publication from the CLF. We will also release a video on the helixray youtube video channel. In the longer term, we expect to design new compounds, and any with suitable properties will be tested for cytotoxicity. If such compounds show promise, we will seek partners to develop that aspect of the work.


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Description Photo-dynamic therapy, a form of treatment for a number of conditions including several cancers and psoriasis, is an area of intense research activity. This treatment strategy uses light to activate a drug in a specific area of the body and can reduce the side effects observed through conventional anti-cancer treatments. Now a group of scientists working in Reading and Dublin have published a new way of finding out how such compounds work at a fundamental level, the information from which can be used to improve anti-cancer drug development. It is really difficult to observe such fast processes in living cells, but the much simpler environment of a DNA crystal has enabled the team to watch the initial crucial step in great detail. The new method is published today (19 October) in the leading chemical journal Nature Chemistry. The crystals contain a ruthenium compound which is bound to a short piece of DNA. This class of compound is used in DNA sensing and is of interest to the pharmaceutical industry for cancer treatment. The researchers found that by using infrared radiation, they could get a snapshot of the extremely fast process - which occurs in half a billionth of a second - that takes place when light is shone on the crystals. This activates the compound, making it cause damage to DNA. This research was carried out using two UK central research facilities: the laser facilities in the Central Laser laboratory of the Science and Technology Facilities Council (STFC) and Diamond Light Source, the UK national synchrotron facility. Dr Susan Quinn, from the School of Chemistry at University College Dublin is the lead author of the study. She said: "These results are very exciting as they demonstrate the ability to follow the flow of electrons from DNA to a molecule whose exact position is known and this is an enormous advantage in the study of the early events that lead to DNA damage." Professor Christine Cardin, from the University of Reading, is a nucleic acid crystallographer who led the UK team, including co-author Dr James Hall, and who has received major funding from BBSRC in support of this work. She said: "This work is an exciting step in helping us to understand DNA damage. Among other things, the insights from this study will feed into the development of new drugs that target cancerous tissue, without damaging healthy tissue around it. "This paper is one result of a longstanding collaboration with Professor John Kelly in Trinity College Dublin, and an excellent example of multidisciplinary international collaboration. "It also highlights the benefits of combining the facilities of Diamond Light Source, where all the crystallography has been carried out, with the equipment and expertise available in the adjacent Central Laser Facility of the STFC." A key element of the funding for the collaboration has been provided by the Royal Irish Academy-Royal Society exchange programme, running since 2008 between Trinity College Dublin and the University of Reading.
Exploitation Route As explained in the Chemistry World article, and also in the BBSRC Business magazine (edition Winter 2016, p16) by defining the first step in DNA damage, we hope to inspire the developement of new drugs for photodynamic therapy, which can target cancerous tissue without damaging the healthy surrounding tissue. By defining carefully the criteria for the earliest step to take place, we are making an important contribution to drug design.
Sectors Chemicals,Pharmaceuticals and Medical Biotechnology