DESIGNED DNA CRYSTALS

设计的 DNA 晶体

• 批准号: 8170586
• 负责人: JENS J BIRKTOFT
• 金额: $ 1.05万
• 依托单位: BROOKHAVEN SCIENCE ASSOC-BROOKHAVEN LAB
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8170586
• 关键词: Binding; Biological; Color; Complex; Computer Retrieval of Information on Scientific Projects Database; Crystallization; DNA; Data Collection; Dyes; Funding; Goals; Grant; Institution; Length; Link; Memory; Molecular; Nature; Optics; Peptides; Proteins; Publishing; Reporting; Research; Research Personnel; Resolution; Resources; Roentgen Rays; Source; Structure; System; Time; United States National Institutes of Health; Vertebral column; base; design; macromolecule; nanoparticle; peptidomimetics; programs; scaffold; synthetic construct

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Our research program is designed to utilize new types of macromolecular building blocks based on branched DNA, as the basis of specifically designed crystalline arrangements 3D structural motifs. The ultimate goals are to provide macromolecular scaffoldings, capable of binding, orienting and juxtaposing a variety of molecules, from biological macromolecules to organic conductors and optical memory components. We proposed and succeeded in determining structures of such a designed 3D system, the tensegrity triangle. This structure is a robust motif with three-fold rotational backbone symmetry, consisting of three helices that are directed in linearly independent directions, i.e., their helix axes do not all share the same plane. The helices are connected pair-wise by three Holliday-like crossover points, so as to produce an alternating over-and-under motif. Recently, we reported [Zheng et al. Nature 461, 74-77 (2009)] the X-ray crystal structure to 4 ¿ of a tensegrity triangle containing a single molecular species, comprised of three helical domains, each containing two double helical turns. Each triangle is centered on a vertex of a rhombohedron, creating a large cavity. Our more recent efforts have been directed in several different directions, all based on the structure of the published two-turn triangle. (A) We have been successful in increasing the numbers of helical turns in the triangle from 2 to 3 and 4 and in determining their crystal structures. While the refined structures all have the structural parameters predicted from their designs, the resolutions of the crystals obtained decrease with the increase in the number of helical turns. We are exploring the effect of changing the length of sticky ends that link the triangles, and also the impact of using natural DNA, rather than synthetic DNA, on the resolution of the crystals. (B) We are attempting to incorporate guest molecules into the internal cavities of the crystal structures; the cavities of the three structures with different length edges (2, 3 and 4 turns) are ~100 nm3, ~375 nm3 and ~1000 nm3. The guest species range from proteins, peptides, and peptidomimetics to dyes, metallic nanoparticles and segments of DNA. Isomorphous crystals have been obtained of such complexes both by co-crystallization and soaking. A report describing the crystal structure of a DNA crystal containing two distinct triangles programmed to crystallize according to the design is ready for submission. In addition, we have also demonstrated that the colors of the crystals can be controlled by the covalent attachment of dye molecules to the different molecules. As a rule our crystals are weakly diffracting, display a high degree of mosaicity and frequently appear in space group P1. Consequently the crystals require long exposure times, small oscillation angles and optimally data collection over 360 degrees.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 我们的研究计划旨在利用基于分支 DNA 的新型大分子构建模块，作为专门设计的晶体排列 3D 结构图案的基础。最终目标是提供大分子支架，能够结合、定向和并置各种分子，从生物大分子到有机导体和光学记忆组件。 我们提出并成功确定了这种设计的 3D 系统的结构，即张拉整体三角形。 该结构是具有三重旋转主干对称性的稳健基序，由沿线性独立方向定向的三个螺旋组成，即它们的螺旋轴并不全部共享同一平面。 螺旋通过三个霍利迪式交叉点成对连接，从而产生交替的上下图案。最近，我们报道了[Zheng et al. Nature 461, 74-77 (2009)] 包含单一分子种类的张拉整体三角形的 X 射线晶体结构，由三个螺旋域组成，每个螺旋域包含两个双螺旋圈。 每个三角形都以菱面体的一个顶点为中心，形成一个大空腔。 我们最近的努力针对几个不同的方向，所有这些都基于已发布的两匝三角形的结构。 (A) 我们成功地将三角形的螺旋圈数从 2 圈增加到 3 圈和 4 圈，并确定了它们的晶体结构。 虽然精细结构都具有根据其设计预测的结构参数，但所获得的晶体的分辨率随着螺旋圈数的增加而降低。我们正在探索改变连接三角形的粘性末端长度的影响，以及使用天然 DNA（而不是合成 DNA）对晶体分辨率的影响。 (B) 我们正在尝试将客体分子纳入晶体结构的内部空腔中；具有不同长度边缘（2、3 和 4 圈）的三种结构的空腔分别为 ~100 nm3、~375 nm3 和 ~1000 nm3。 客体种类包括蛋白质、肽、拟肽、染料、金属纳米粒子和 DNA 片段。 通过共结晶和浸泡均获得了此类配合物的同晶晶体。 一份描述 DNA 晶体晶体结构的报告已准备好提交，该 DNA 晶体包含两个不同的三角形，根据设计进行编程以结晶。 此外，我们还证明晶体的颜色可以通过染料分子与不同分子的共价连接来控制。 通常，我们的晶体衍射较弱，表现出高度的镶嵌性，并且经常出现在 P1 空间群中。 因此，晶体需要较长的曝光时间、较小的振荡角度以及超过 360 度的最佳数据收集。