Probing DNA Radiation Damage by DNA Nanotechnology

电磁或粒子辐射对DNA的损伤是肿瘤放射治疗等过程中发生的关键过程。在放射治疗中，使用诸如卤代核苷之类的放射增敏剂使肿瘤组织对辐射增敏。但是DNA在初级高能辐射中只受到很小程度的破坏。大多数损伤是由低能次级粒子如电子引起的。同时也证实了动能小于12ev的电子可以通过解离电子附着机制破坏DNA。然而，潜在的过程只被很好地理解为简单的模型化合物，如单核碱基。DNA序列对DNA链断裂的影响和放射增敏剂的作用在初步实验中被提出，但由于缺乏合适的分析工具，无法进行系统的研究。在申请人之前的项目中，建立了一种测定特定序列寡核苷酸DNA链断裂率的新技术。该技术的关键方面是目标寡核苷酸序列在DNA折纸纳米结构上的排列和利用原子力显微镜（AFM）检测电子诱导链断裂。通过这种方式，可以在一次辐照实验中比较多个DNA序列，并且可以直接获得链断裂的绝对横截面。在目前的建议中，电子诱导链断裂的横截面将被确定为DNA序列，这些序列被临床建立的和潜在的放射增敏剂修饰。通过与未修饰序列的比较，确定了不同电子能量下的增强因子。研究人员将系统地修改放射增敏剂的分子结构，以揭示损伤机制，并能够促进新型放射增敏剂的开发。此外，还将研究水环境对DNA损伤的影响。金纳米粒子被用作光电子的来源，而光电子会破坏溶液中的DNA。溶剂的影响将一方面通过DNA折纸技术和原子力显微镜测定链断裂率，并与真空实验进行比较来研究。另一方面，利用表面增强拉曼散射可以检测到损伤产物。这样就可以得到关于水环境中辐射引起的化学变化的补充资料。这是在日益复杂的系统中探索损伤机制的一种新方法，最终目标是揭示DNA放射致敏的物理化学基础。

The damage of DNA by electromagnetic or particle radiation is a pivotal process occurring for instance during tumor radiation therapy. In radiotherapy radiosensitizers such as halogenated nucleosides are applied to sensitize the tumour tissue towards radiation. But the DNA is damaged only to a small extent by the primary high-energy radiation. Most of the damage occurs due to low-energy secondary particles such as electrons. In the meantime it is well-established that electrons with kinetic energy below 12 eV can damage DNA via the dissociative electron attachment mechanism. However, the underlying processes are only well-understood for simple model compounds such as single nucleobases. The influence of the DNA sequence on the DNA strand breakage and the effect of radiosensitizers was suggested within preliminary experiments, but could not be investigated systematically due to a lack of suitable analytical tools. In the previous project of the applicant a novel technique for the determination of DNA strand break yields in oligonucleotides of defined sequence was established. The key aspects of this technique are the arrangement of the target oligonucleotide sequences on DNA origami nanostructures and the detection of electron-induced strand breaks by atomic force microscopy (AFM). In this way, multiple DNA sequences can be compared within a single irradiation experiment, and absolute cross sections for strand breakage are directly accessible. In the present proposal cross sections for electron induced strand breakage will be determined for DNA sequences, which are modified with clinically established and potential radiosensitizers. By comparison with non-modified sequences enhancement factors will be determined at different electron energies. The molecular structure of the radiosensitizers will be modified systematically to reveal damage mechanisms and to be able to stimulate the development of novel radiosensitizers. Furthermore, the influence of an aqueous environment on the DNA damage will be studied. Gold nanoparticles are used as a source of photoelectrons, which can damage the DNA in solution. The influence of the solvent will on the one hand be studied by determination of strand break yields using the DNA origami technique and AFM and by comparison with vacuum experiments. On the other hand damage products can be detected using surface-enhanced Raman scattering. In this way complementary information about radiation induced chemical changes in aqueous environment will be obtained. This is a novel approach to explore damage mechanisms in increasingly complex systems and the final goal is to reveal the physico-chemical basis of DNA radiosensitization.