DNA - DNA interactions with atomic detail

描述（由申请人提供）：DNA和RNA缩合是生物学和生物技术的广泛兴趣。这些核酸（NAs）的体内包装能够有效地储存遗传信息；体外包装策略对于治疗方法的发展至关重要，例如，用于非病毒基因传递。DNA双链之间的同电荷吸引是DNA凝聚的重要前体，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钴六胺，到活细胞中发现的更棒状、电荷密度更低的多胺；例如，精胺和亚精胺。尽管RNA对生物学具有同等的重要性，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应用的强烈动机。本提案的总体目标是开发多价离子- na双相相互作用的定量机制，直接导致离子诱导的DNA或RNA吸引。该机制将以基本物理为基础，但将足够详细地解决许多实际问题，如核酸凝聚的序列依赖性和实验观察到的RNA与DNA凝聚行为的显著差异。现有的理论模型强调了产生这些吸引力的离子相关性的必要性，但在原子水平上描述吸引力的机制还没有达成共识。目前的实验方法本身不具备提供各种核酸凝聚现象的原子细节图像的分辨率。为了了解DNA或RNA双链之间的反离子诱导吸引机制，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，以提供必要的分辨率。该方法将基于新型隐式溶剂/显式离子模型（用于速度和采样效率）的大规范蒙特卡罗模拟与微秒时间尺度（用于最高细节水平）的传统显式溶剂分子动力学模拟相结合。我们方法的独特之处在于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对核酸周围离子结合和空间分布的实验测量来验证；这些数据将用于对模型的参数进行微调。小角度x射线散射技术将探测NA双离子大气的结构，而使用紫外光谱的冷凝实验将提供与理论具体预测的额外程度的比较。一旦理论和实验在更简单的系统中达成一致，我们将探索核酸序列和拓扑结构在凝聚中的作用，并从原子的角度理解生物学上重要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.