DNA DESIGNER CRYSTALS -- 3D TRIANGLE, DODECAMER, 16MER

DNA 设计师水晶 -- 3D 三角形、十二角、16MER

• 批准号: 7957298
• 负责人: JENS J BIRKTOFT
• 金额: $ 1.14万
• 依托单位: BROOKHAVEN SCIENCE ASSOC-BROOKHAVEN LAB
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7957298
• 关键词: Behavior; Binding; Computer Retrieval of Information on Scientific Projects Database; Crystallization; Crystallography; DNA; DNA Fingerprinting; Diffusion; Funding; Gel; Grant; Heating; High Pressure Liquid Chromatography; Institution; Laboratories; Length; Light; Methods; Modification; Periodicity; Process; Protocols documentation; Research; Research Personnel; Resolution; Resources; Roentgen Rays; Source; Structure; Suggestion; Synchrotrons; Temperature; United States National Institutes of Health; Work; X ray diffraction analysis; X-Ray Diffraction; design; improved; macromolecule; melting; scaffold; vapor

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. We have prepared three types of DNA crystals. The first one is called triangle. The space group is R3. Resolution we got is about 5A. The second one is dodecamer DNA crystal. The space group is C2. The resolution we can get for it is about 3A. The third one is 16mer DNA crystal. The space group is P43. The resolution is about 3.1A. Our group has designed a variety of DNA motifs expected to form 3D lattices. Ultimately, the applications of 3D DNA lattices include providing scaffolding that is capable of binding and orienting a variety of species. Crystals of macromolecules and notoriously difficult to grow, and the process of crystallization relies heavily on pure luck. DNA cages could act as a host to these molecules, aligning them in a periodic fashion necessary for X-ray analysis. The 3D designs pursued in our laboratory are predicated on Watson-Crick complementarities. In order to confirm the formation of 3D DNA lattices, we purify the strands corresponding to those designs and self-assemble them into the desired motifs. It will attempt to form crystalline lattices with the DNA motifs. When crystals appear, they will be characterized by X-ray diffraction methods. Derivatized crystals will be prepared, and the structures solved when the resolution is high enough to warrant such studies. A 3D triangle was made in this work involved incorporating DX molecules to replace the double helices. Crystals of this motif were grown using a combination of vapor diffusion and a heating protocol. They ranged from 100 to 200um in length. A two-tile version of the 3D DX triangle has been designed and the AFM evidence supports its formation. So far, we have completed the DLS characterization of the melting behaviors of the aggregates so as to determine proper temperature ranges and preferred lattice periodicities and to establish a relationship between the melting temperatures established by the DLS and the proper temperature at which to attempt crystallization. In our lab we also tried HPLC purification followed by gel purification. The improved resolution on some simple version encourages us to apply HPLC purification on this motif. Also followed Dr. Sherman¿¿¿¿¿"s suggestion, we modified the motif design to make sure the structure is in less strain. Hopefully, during this course, we would have crystals from this new modification.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 我们已经制备了三种类型的DNA晶体。 第一个叫做三角形。空间群为R3。分辨率约为5A。 第二种是十二聚体DNA晶体。空间群为C2。我们可以得到的分辨率约为3A。 第三种是16聚体DNA晶体。空间群为P43。分辨率约为3.1A。 我们的团队已经设计了各种各样的DNA基序，有望形成3D晶格。最终，3D DNA晶格的应用包括提供能够结合和定向各种物种的支架。大分子的晶体是出了名的难以生长，结晶的过程在很大程度上依赖于纯粹的运气。DNA笼可以作为这些分子的宿主，以X射线分析所需的周期性方式排列它们。我们实验室追求的3D设计是基于沃森-克里克互补性。为了确认3D DNA晶格的形成，我们纯化了与这些设计相对应的链，并将它们自组装成所需的基序。它将尝试形成带有DNA基序的晶格。当晶体出现时，将通过X射线衍射方法对其进行表征。将制备衍生晶体，当分辨率足够高以保证此类研究时，解析结构。 在这项工作中制作了一个3D三角形，涉及将DX分子取代双螺旋。使用蒸汽扩散和加热方案的组合生长该图案的晶体。它们的长度从100到200 μ m不等。一个两瓦版本的三维DX三角形的设计和AFM证据支持其形成。到目前为止，我们已经完成了DLS表征的聚集体的熔融行为，以确定适当的温度范围和优选的晶格周期性，并建立由DLS建立的熔融温度和尝试结晶的适当温度之间的关系。 在我们的实验室中，我们还尝试了HPLC纯化，然后进行凝胶纯化。在一些简单的版本上改进的分辨率鼓励我们对该基序应用HPLC纯化。也遵循谢尔曼博士的建议，我们修改了主题设计，以确保结构是在较少的应变。希望，在这门课上，我们能从这个新的修改中得到晶体。