ETNA - Expansion of the Time domain in Nucleic Acid crystallography

Lead Research Organisation: University of Reading
Department Name: Chemistry

Abstract

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.

Publications

10 25 50
 
Title Cover design for Angewandte Chemie 
Description Cover design for to accompany our Angewandte Chemie article designed by Drs James Hall and Paraic Keane (the PDRAs on the BBSRC grants) 
Type Of Art Artwork 
Year Produced 2015 
Impact Greater visibility and (in the future) increased citation rate for this study. 
URL http://onlinelibrary.wiley.com/doi/10.1002/anie.201582961/abstract
 
Title Cover for Nature Chemistry 
Description Cover for December 2015 issue of Nature Chemistry (in which our paper was published) was designed by Dr James Hall. 
Type Of Art Artwork 
Year Produced 2015 
Impact Greater visibility for our work across digital and social media. 
URL http://www.nature.com/nchem/journal/v7/n12/covers/index.html
 
Title Graphic in University Health Exhibition 
Description Graphic in University Health Exhibition 
Type Of Art Image 
Year Produced 2015 
Impact Awareness of our research has increased 
 
Title Helixray video channel 
Description The helixray video channel was established by Christine Cardin in 2012 to promote the understanding of the crystal structures used in these and other studies. To date it has had over 43,000 views from all around the world. It makes the concepts behind our work available even to those who have no access to the original literature. 
Type Of Art Film/Video/Animation 
Year Produced 2012 
Impact As above, over 43,000 views and many new contacts. 
URL https://www.youtube.com/channel/UCQnXJvvIRgkhRArO1dnZY8Q?view_as=public
 
Description For the first time we showed directly the first step in DNA damage, the oxidation of guanine.
Exploitation Route Photodynamic therapy
Sectors Chemicals,Pharmaceuticals and Medical Biotechnology

URL http://www.bbsrc.ac.uk/news/health/2015/151019-pr-new-technique-offers-light-activated-therapies
 
Description Helixray video channel created to demonstrate nucleic acid and other macromolecular structure.
First Year Of Impact 2012
Sector Education
Impact Types Cultural

 
Description Diamond-EPSRC studentship for Kane McQuaid
Amount £71,110 (GBP)
Organisation Diamond Light Source 
Sector Academic/University
Country United Kingdom
Start 10/2015 
End 03/2019
 
Description UROP summer studentship
Amount £1,600 (GBP)
Organisation University of Reading 
Sector Academic/University
Country United Kingdom
Start 06/2016 
End 09/2016
 
Title Temperature controlled cell for Ultrafast measurements 
Description Development of a 3D-printed temperature controlled sample holder for use in transient infra-red spectroscopy. 
Type Of Material Improvements to research infrastructure 
Year Produced 2016 
Provided To Others? Yes  
Impact We have been able to examine the mechanism of DNA damage under a number of different temperatures. The data are currently being processed but it is expected that we will be able to comment on the fundamental mechanism of electron transfer. 
 
Description APS beamtime 
Organisation University of Hamburg
Department Centre for Ultrafast Imaging
Country Germany 
Sector Academic/University 
PI Contribution Supply of ruthenium compounds for XAS study by Dr Nils Huse. e.g. [Ru(TAP)2dppz]2+
Collaborator Contribution Award of beamtime at the Advanced Photon Source, Chicago, for Transient XAS measurements on these compounds.
Impact Beamtime in summer 2016
Start Year 2016
 
Description CruX 
Organisation Trinity College Dublin
Country Ireland 
Sector Academic/University 
PI Contribution Crystal growth and structure determination for ultrafast kinetics using the ULTRA facility of the CLF. PI for this programme.
Collaborator Contribution Data measurement and analysis from the ULTRA laser
Impact Award of ETNA project by BBSRC (BB/M004635/1) - starting date 1.10.2014.
Start Year 2013
 
Description CruX 
Organisation University College Dublin
Country Ireland 
Sector Academic/University 
PI Contribution Crystal growth and structure determination for ultrafast kinetics using the ULTRA facility of the CLF. PI for this programme.
Collaborator Contribution Data measurement and analysis from the ULTRA laser
Impact Award of ETNA project by BBSRC (BB/M004635/1) - starting date 1.10.2014.
Start Year 2013
 
Description Neutron crystallography beamtime 
Organisation Oak Ridge National Laboratory
Country United States 
Sector Public 
PI Contribution Growth of crystals for neutron diffraction experiments
Collaborator Contribution Forthcoming beamtime awarded in May 2016
Impact None as yet
Start Year 2016
 
Description Article in Chemistry World October 2015 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact 634 Facebook 'likes' and a follow-up article commissioned from the same author (Andy Extance) by the RSC, on pump-probe methodology. DR Extance visited Diamond Light Source and the Central laser facility while the team were at work to see the techniques and the team in action.
Year(s) Of Engagement Activity 2015
URL http://www.rsc.org/chemistryworld/2015/10/observing-dna-damage-light-free-radical
 
Description BBSRC Business magazine Winter 2016 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Whole page article 'New technique offers a window into light-activated therapies'.
Year(s) Of Engagement Activity 2016
URL http://www.bbsrc.ac.uk/news/health/2015/151019-pr-new-technique-offers-light-activated-therapies/
 
Description SET for Britain 2016 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact Dr James Hall was selected, and was the only representative from the University of Reading, to present a poster to MPs, specifically the MP for Reading West, Rob Wilson, and that for Wokingham, John Redwood. the event was covered by the Royal Society of Chemistry among others.
Year(s) Of Engagement Activity 2016
URL http://www.setforbritain.org.uk/2016event.asp