DNA - DNA interactions with atomic detail

描述(申请人提供)：DNA和RNA缩合在生物学和生物技术中都引起了广泛的兴趣。这些核酸(Nas)的体内包装使遗传信息的有效存储成为可能；体外包装策略对于开发治疗药物至关重要，例如用于非病毒基因传递。DNA双链体之间的同电荷吸引是DNA缩合的重要前体，可以由具有明显不同物理性质的多价离子诱导，从球形三价无机离子六胺钴到活细胞中发现的更棒状、电荷密度较低的多胺，如精胺和亚精胺。尽管RNA对生物学同样重要，但人们对双链RNA缩合几乎一无所知。最近发现的RNA干扰(RNAi)过程是确定短RNA双链是否可以包装用于治疗的强烈动机。这一提议的主要目标是开发一种直接导致离子诱导的DNA或RNA吸引的多价离子-NA双链相互作用的定量机制。这一机制将有基本的物理基础，但将足够详细地解决许多实际问题，如核酸缩合的序列依赖性，以及实验观察到的RNA和DNA缩合行为的显著差异。现有的理论模型强调了离子关联产生这些引力的必要性，但在原子水平上描述这种引力的机制还没有达成共识。目前的实验方法本身并不足以提供各种核酸凝聚现象的原子细节图像。为了了解反离子诱导DNA或RNA双链之间吸引的机制，我们将开发并实验测试一种新的方法来模拟核酸的离子环境，该方法提供了必要的分辨率。该方法将基于新的隐式溶剂/显式离子模型的宏正则蒙特卡罗模拟(对于速度和采样效率)与传统的微秒时间尺度的显式溶剂分子动力学模拟(对于最高级别的细节)相结合。我们方法的显著特点是计算模型将与实验紧密结合。多价离子-NA相互作用的预测将通过实验测量离子与核酸的结合和周围的空间分布来验证；这些数据将用于微调模型的参数。小角X射线散射技术将探测NA双链离子大气的结构，而使用UV光谱的凝聚实验将提供与理论特定预测的额外比较程度。一旦理论和实验在更简单的系统上达成一致，我们将探索核酸序列和拓扑在缩合中的作用，并从原子角度理解具有生物重要性的多离子精胺和亚精胺如何促进核酸片段之间的吸引。

DESCRIPTION (provided by applicant): DNA and RNA condensation is of broad interest in both biology and biotechnology. The in vivo packaging of these nucleic acids (NAs) enables the efficient storage of genetic information; strategies for in vitro packaging are crucial for development of therapeutics, e.g., for non-viral gene delivery. Like-charge attraction between DNA duplexes is an essential precursor of DNA condensation and can be induced by multivalent ions with distinctly different physical properties, ranging from the spherical trivalen inorganic ion, cobalt hexa-amine, through the more rod-like, less charge-dense polyamines found in living cell; e.g., spermine and spermidine. Despite an equal importance of RNA to biology, almost nothing is known about double-stranded RNA condensation. The recent discovery of the RNA interference (RNAi) process is strong motivation for determining whether short RNA duplexes can be packaged for therapeutic applications. The broad goal of this proposal is to develop a quantitative mechanism for multivalent ion-NA duplex interactions that leads directly to ion-induced DNA or RNA attraction. This mechanism will have basic physics at its foundation, yet will be detailed enough to address many practical questions such as sequence dependence of nucleic acid condensation and the striking differences in RNA vs. DNA condensation behavior observed experimentally. Existing theoretical models stress the necessity of ion correlations for generation of these attractive forces, yet no consensus exists describing the mechanism for the attraction at atomic level. Current experimental methods alone do not have the resolution to deliver the atomically detailed picture of the diverse nucleic acid condensation phenomena. To understand the mechanism of counterion-induced attraction between DNA or RNA duplexes, we will develop and experimentally test a new approach to modeling ionic environments of nucleic acids that provides the necessary resolution. The approach will integrate grand canonical Monte Carlo simulations based on novel implicit solvent/explicit ion models (for speed and sampling efficiency) with the traditional explicit solvet molecular dynamics simulations on microsecond time scales (for the highest level of detail). The distinctive feature of our approach is that the computational models will be developed in tight integration with the experiment. Predictions of multivalent ion-NA interactions will be validated by experimental measurement of ion association to and spatial distribution around nucleic acids; the data will be used to fine- tune parameters of the model. Small angle X-ray scattering techniques will probe the structure of NA duplex ionic atmospheres, while condensation experiments using UV spectroscopy will provide additional degrees of comparison with specific predictions of the theory. Once agreement between theory and experiment on simpler systems is reached, we will explore the role of nucleic acid sequence and topology in condensation, and develop an atomistic understanding of how the biologically important polyions spermine and spermidine facilitate attraction between nucleic acid fragments.