ETNA - Expansion of the Time domain in Nucleic Acid crystallography

1980年，DNA的标志性双螺旋结构在单晶体结构中得到了证实，当时有可能使DNA的小合成片段有序，并将它们纯化到可以结晶的状态。在那个阶段，由于x射线源的功率仍然相当有限，因此需要相对较大的晶体，大约0.5毫米大小。目前的技术，比如钻石光源公司的技术，意味着我们可以在如此小的DNA片段中精确地看到单个碳原子，即使是在室温下的晶体中，也可以在水溶液中看到。我们可以在包括室温在内的一系列温度下，在不同程度的水合作用下，在小到5微米大小的晶体上做这件事。通过使用更短的波长和更大的晶体（0.1毫米左右），我们也可以测量非常精确的原子位置，因为短波长实际上扩展了我们可以看到的细节（提高了分辨率），甚至可能扩展到直接观察氢原子。在相同的时间尺度上，超快激光测量已经发展到可以用来在皮秒时间尺度上跟踪分子光激发后电子的运动，并且可以监测光吸收的极其微小的差异。这些微小的差异与分子激发态中电子分布的差异有关。例如，最近这项技术已被用于研究光敏剂引起的DNA损伤的最早步骤，以及类似于暴露在太阳有害的UVA和UVB射线下造成的直接损伤。一个这样的系统是由钌聚吡啶“光开关”和相关复合物形成的。我们最近首次发表了这些化合物与DNA双链结合的晶体结构，因此在研究这些系统方面处于独特的有利地位。我们现在建议将这两种强大的技术结合起来，不仅可以追踪DNA的快速激发和可能的光损伤，还可以精确定位这种激发发生的位置。我们将利用我们的专业知识来制备合适的晶体，用于光敏和直接DNA损伤。为了扩展这个最初的想法（我们已经有足够的初步数据来确信我们的样品制备方法会产生有用的结果），我们将对其中一些系统进行选择性化学改性。DNA碱基被描述为“大自然的防晒霜”，因为它们似乎是为了抵抗直接照射而被选择的，但微小的修饰（如用硫羰基硫类似物取代鸟嘌呤6-羰基）会大大延长光化学寿命，相应地，DNA损伤的可能性更大，这确实是一些用作光动力治疗中致敏剂的化合物的已知副作用。目前正在建设的LIFEtime仪器的新超快技术将被我们用来监测这些损坏过程中的连续步骤。理想情况下，我们希望能够追踪众所周知的损伤产物，如8-氧鸟嘌呤的形成，在这个实验中，我们将使用已知的通过这种机制造成损伤的光敏剂。其中一种含金属的光敏剂是铼络合物，它与钌络合物具有一些共同的化学特征。晶体学和超快动力学测量的结合应该给我们一个光诱导DNA损伤过程的“电影”。

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.