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

9304467 HO任何结晶学实验的第一步，也是最关键的一步，就是获得能绕射X射线的单晶。本方案中的实验试图系统地开发用于结构研究的A-、B-和Z-DNA晶体的生长规则。影响寡核苷酸在单晶中的生长模式和最终结构的主要因素有两个：1)DNA的构象；2)保持分子在晶体对称排列中的力(即晶体堆积力)。何博士此前曾证明，DNA在晶体中的构象取决于它在结晶溶液中的构象。这反过来又受到结晶溶液条件和DNA结晶顺序的影响。对于Z-DNA，何博士之前曾通过估计特定序列形成Z-DNA的倾向来预测结晶条件。Z-DNA的稳定性是根据给定序列在B构象和Z构象中的水合自由能之差来计算的。何博士建议继续这项工作，扩大计算范围，以包括对Z-DNA形成有立体抑制作用的序列的Z-DNA稳定性的经验估计。此外，同样的方法也可以用来预测DNA A型结晶的条件。为了使B-DNA结晶，需要注意只稳定这种右旋构象的特定序列要求。此外，还将讨论晶体堆积力的问题，它们如何影响结晶，以及一旦进入晶体，它们如何影响寡核苷酸的结构。将进行实验以确定对这些能量的焓和熵的贡献，并比较空间和疏水对晶格内分子间相互作用的影响。40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。这种一般结构通过定义细胞内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的结构被证明比最初怀疑的更加多变，出现了新的弯曲和扭曲，可能会作为额外的信号来控制转录和复制等基本细胞功能。再一次，X射线衍射在发现这些新的和不同的结构方面发挥了重要作用。然而，这些最近的研究利用了短DNA片段(寡核苷酸)的单晶，这给了我们特定DNA序列在原子水平上的结构。这些研究的主要障碍一直是生长适合于结构确定的单晶的能力。这一过程通常更多地被视为一门艺术，而不是一门量化科学。这是令人惊讶的，因为结晶学被认为是一门非常严格的科学。何博士建议进行一些研究，试图定义使各种构象中的DNA序列结晶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。

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.