Mechanisms for Radiation Damage to DNA: LET Effects

描述(申请人提供)：这项研究的目标是阐明变化的线性能量转移(LET)辐射对DNA造成辐射损伤的基本机制。我们的DNA辐射损伤综合模型描述了从DNA离子自由基和激发态的初始形成，到空穴和电子转移，再到糖自由基的形成，最后到分子产物的事件，我们将在每一步进行测试，以阐明导致DNA辐射损伤的基本过程。这些研究是在强调辐射直接影响的条件下进行的，将使用磁共振光谱、密度泛函理论和产物分析技术以及伽马和回旋重离子束照射。有三个目标：第一个目标将解决空洞引起的DNA辐射损伤中的几个主要悬而未决的问题。这一目标将专门使用C-8氚标记的定义序列寡聚物来利用我们实验室最近的一项突破，该突破使我们能够区分C-8氚标记的嘌呤碱基(鸟嘌呤或腺嘌呤)和未标记的位置上的孔(阳离子自由基)。我们还发现，C-8标记可以区分鸟嘌呤和腺嘌呤的阳离子自由基和它们的去质子化形式。通过这些进展，我们将发现：a.空穴局部化的碱基序列依赖性，b.dsDNA中特定位置的鸟嘌呤和腺嘌呤阳离子自由基的质子化状态，c.空穴激发时碱基到碱基和碱基到糖转移的程度。我们的第二个目标是识别自由基的形成和跟踪结构，作为让离子束照射的DNA的函数。我们将通过ESR光谱来识别自由基，并确定它们的空间分布和聚集性，作为辐射沿辐射轨迹的LET的函数。尤其重要的是对最近发现的由糖磷酸主链断裂引起的即刻断链自由基的LET依赖性的研究。径迹核心中自由基形成和聚集的性质与理解DNA中最重要的损伤--不可修复的倍增损伤部位的形成有关。我们的最终目标将使用理论计算来进一步测试和确认上述研究中提出的分子机制。尤其重要的将是用TD-DFT理论来处理碱性离子自由基的激发态，这些自由基现在与DNA链断裂有关，并且随着辐射LET的增加而变得更加重要。我们相信，这一努力将使我们能够对生物医学研究中重要的基本辐射过程建立新的见解。 公共卫生相关性：这项研究的目标是通过阐明不同线性能量转移(LET)辐射对DNA损伤的基本机制，开发一个全面的DNA辐射损伤模型。我们的DNA辐射损伤模型描述了从DNA离子自由基和激发态的初始形成，到空穴和电子转移，再到糖自由基的形成，最后到分子产物的事件，我们将在每一步进行测试，以阐明导致DNA辐射损伤的基本过程。这些研究是在强调辐射直接影响的条件下进行的，将使用伽马和回旋重离子束照射、磁共振光谱、密度泛函理论和产品分析技术，并将解决对生物医学研究重要的DNA辐射损伤中的主要悬而未决的问题。

DESCRIPTION (provided by applicant): The goal of this research is to elucidate fundamental mechanisms of radiation damage to DNA by radiations of varying linear energy transfer (LET). Our comprehensive model for DNA radiation damage that describes events from the initial formation of DNA ion radicals and excited states, to hole and electron transfer, to sugar radical formation and finally to molecular products will be tested at each step to clarify the fundamental processes resulting in DNA radiation damage. These studies, which are performed under conditions that emphasize the direct effect of radiation, will employ magnetic resonance spectroscopies, density functional theory and product analysis techniques as well as gamma and cyclotron heavy ion beam irradiations. There are three aims: The first aim will address several of the major unanswered questions in DNA radiation damage induced by holes. This aim will employ specifically C-8 deuterium labeled defined sequence oligos to exploit a recent breakthrough in our laboratory that allows us to distinguish a hole (cation radical) at a C-8 deuterium labeled purine base (guanine or adenine) from an unlabeled site. We have also found that the C-8 labeling allows the distinction of the guanine and adenine cation radicals from their deprotonated forms. With these developments we will find: a. the base sequence dependence of hole localization, b. the protonation states of guanine and adenine cation radicals at specific sites in dsDNA, c. the extent of base-to- base versus base-to-sugar transfer on hole excitation. Our second aim will identify radicals formed and track structure as a function of LET in ion beam irradiated DNA. We will identify radicals via ESR spectroscopy and ascertain their spatial distribution and clustering as a function of the LET of the radiation along the radiation track. Especially important will be a study of the LET dependence of recently discovered prompt strand break radicals that result from cleavage of the sugar phosphate backbone. The nature of the radical formation and clustering in the track core is pertinent to understanding the formation of the most important lesion in DNA the unrepairable multiply damaged site. Our final aim will employ theoretical calculations to further test and confirm molecular mechanisms proposed in the above studies. Especially significant will be treatment by TD- DFT theory of excited states of base ion radicals which are now implicated in DNA strand breaks and become more significant as the LET of the radiation increases. We believe this effort will allow us to establish new insights into fundamental radiation processes important for biomedical research. PUBLIC HEALTH RELEVANCE: The goal of this research is to develop a comprehensive model of DNA radiation damage by elucidating fundamental mechanisms of damage to DNA by radiations of varying linear energy transfer (LET). Our model for DNA radiation damage that describes events from the initial formation of DNA ion radicals and excited states, to hole and electron transfer, to sugar radical formation and finally to molecular products will be tested at each step to illuminate the fundamental processes resulting in DNA radiation damage. These studies, which are performed under conditions that emphasize the direct effect of radiation, will employ gamma and cyclotron heavy ion beam irradiations, magnetic resonance spectroscopies, density functional theory and product analysis techniques and will address major unanswered questions in DNA radiation damage important to biomedical research.