Predicting the Crystalization of A-, B-, and Z-DNA

预测 A、B 和 Z-DNA 的结晶

• 批准号: 9304467
• 负责人: Pui Ho
• 金额: $ 30万
• 依托单位: Oregon State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 1997-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9304467&HistoricalAwards=false
• 关键词: Predicting; Crystalization; DNA

9304467 Ho The first and most critical step in any crystallographic experiment is to obtain a single crystal that diffracts x-rays. The experiments in this proposal attempts to systematically develop rules for growing crystals of A-, B-, and Z-DNA for structural studies. There are two primary factors that affect the growth pattern and the ultimate structure of an oligonucleotide in a single crystal: 1) the conformational of the DNA and 2) the forces holding the molecules in the symmetric arrangement of the crystal (i.e. the crystal packing forces). Dr. Ho has previously show that the conformation of the DNA in a crystal is dependent on its conformation in the crystallization solutions. This, in turn, is affected by the solution conditions for crystallization and the sequence of the DNA being crystallized. For Z-DNA, Dr. Ho had previously predicted crystallization conditions by estimating the propensity of a particular sequence to form Z-DNA. The stability of Z-DNA is calculated as the difference in the hydration free energy for a given sequence in the B- versus Z-conformations. Dr. Ho proposes to continue this work by extending the calculations to include empirical estimates of Z-DNA stability for sequences that have steric inhibitions to Z-DNA formation. Furthermore, this same approach can be used to predict the conditions for crystallizing the A- form of DNA. To crystallize B-DNA, attention will be paid to specific sequence requirements that stabilizes only this right- handed conformation. In addition, the problem of crystal packing forces, how they affect crystallization and how they affect the structure of an oligonucleotide once in the crystal will be addressed. Experiments will be carried out to determine the enthalpic versus the entropic contributions to these energies, and to compare steric versus hydrophobic effects on the intermolecular interactions within the crystal lattice. %%% The structure of the DNA duplex was first determined forty y ears ago through x-ray diffraction studies on DNA fibers. This general structure launched a new era in the biological sciences by defining the underlying molecular principles of genetic processes in the cell. Since that time, the structure of DNA has been shown to be even more variable than originally suspected, taking new bends and twists that may act as additional signals for controlling such basic cellular functions as transcription and replication. Again x-ray diffraction has played a major role in the discovery of these new and different structures. These recent studies, however, utilize single crystal of short DNA fragments (oligonucleotides), which give us structures of specific DNA sequences at the atomic level. The primary hurdle to these studies has been the ability to grow single crystals that are suitable for structure determinations. This process is generally treated more as an art rather than as a quantitative science. This is surprising since crystallography is perceived as a very exacting science. Dr. Ho proposes studies that attempt to define the intrinsic and extrinsic conditions required to crystallize DNA sequences in the various conformations. This includes the design of the sequence for crystallization, and the types and amount of salts and alcohols that need to be added to these sequences to help stabilize a specific DNA conformation.

[304467]在任何晶体学实验中，第一步也是最关键的一步是获得能衍射x射线的单晶。本提案中的实验试图系统地发展用于结构研究的A-， B-和Z-DNA晶体的生长规则。有两个主要因素影响单晶中寡核苷酸的生长模式和最终结构：1)DNA的构象和2)使分子保持晶体对称排列的力（即晶体填塞力）。何博士先前已经证明，晶体中DNA的构象取决于其在结晶溶液中的构象。这反过来又受到结晶的溶液条件和被结晶的DNA序列的影响。对于Z-DNA， Ho博士之前通过估计特定序列形成Z-DNA的倾向来预测结晶条件。Z-DNA的稳定性计算为B-构象与z -构象中给定序列的水化自由能之差。Ho博士建议通过扩展计算来继续这项工作，以包括对Z-DNA形成具有空间抑制的序列的Z-DNA稳定性的经验估计。此外，同样的方法可以用来预测DNA A-型结晶的条件。为了使B-DNA结晶，将注意特定的序列要求，只稳定这种右手构象。此外，晶体填充力的问题，它们如何影响结晶，以及它们如何影响晶体中寡核苷酸的结构。实验将确定焓和熵对这些能量的贡献，并比较晶格内分子间相互作用的空间和疏水效应。四十年前，通过对DNA纤维的x射线衍射研究，首次确定了DNA双链的结构。这种总体结构通过定义细胞遗传过程的基本分子原理，开启了生物科学的新纪元。从那时起，DNA的结构已经被证明比最初怀疑的更加多变，呈现出新的弯曲和扭曲，这些弯曲和扭曲可能作为控制转录和复制等基本细胞功能的额外信号。x射线衍射在发现这些新的和不同的结构中再次发挥了重要作用。然而，这些最近的研究利用短DNA片段（寡核苷酸）的单晶，在原子水平上为我们提供了特定DNA序列的结构。这些研究的主要障碍是培养适合于结构测定的单晶的能力。这个过程通常被视为一门艺术，而不是一门定量科学。这是令人惊讶的，因为晶体学被认为是一门非常严格的科学。何博士建议进行研究，试图确定以各种构象结晶DNA序列所需的内在和外在条件。这包括结晶序列的设计，以及需要添加到这些序列中以帮助稳定特定DNA构象的盐和醇的类型和数量。