Self-assembled DNA crystals as scaffolds for macromolecules

第一部分：非技术概述：自然利用自组装将分子组织成功能材料，生物细胞是这种方法巨大潜力的主要例子。DNA和RNA是构建定制自组装系统最有前途的两个分子，因为它们以可编程的方式编码信息，所以许多独立的链可以被设计成以可预测的方式聚集在一起。这些核酸的分子性质(如尺寸、几何形状和刚性)也是众所周知的，它们可以形成分支连接，使它们能够在2D和3D空间中组装。基于核酸的纳米技术的一个关键目标是构建具有可编程空隙的3D晶体，以容纳各种客体分子。该项目旨在首次证明，通过将RNA或蛋白质等分子与构成晶格的链条捆绑在一起，可以在这些晶体中进行特定的定位。第一个目标是将RNA引入晶体，既是为了看看它如何改变晶格的组装，也是为了解决像适配子这样的小的、未知的RNA基序的结构。在第二个目标中，蛋白质将附着在晶体上，不是为了解决它们的结构，而是为了创建这些分子的密集3D阵列(例如，出于催化原因，通过结合酶)。最后，该项目的第三个目标是创造纳米晶体(大约比通常获得的晶体小10亿倍)，携带小干扰RNA(SiRNA)或功能蛋白质。这些晶体可以用来有效地将这些货物运送到细胞中，因为它们可以携带极高的密度。综上所述，该提案的目标将创建一种新的3D支架系统，用于解决RNA结构、附着蛋白质以制造催化材料，并更高效地将重要的功能分子输送到细胞中。该项目还将产生重大的社会和教育影响，为K-12教育开发一个在线课程和教师培训，并开发一个新的自我组装在线游戏，以探索这些系统如何工作。该计划将吸引本科生、研究生和未被充分代表的少数族裔学生在科学、技术、工程和数学(STEM)领域获得知识和进行研究，并帮助开发一门新课程，向广泛的学生教授纳米技术。第2部分：技术概述本项目的目标是使用自组装的3D DNA晶体作为功能性大分子支架，可以固定客体分子，如RNA和蛋白质。这些DNA晶体在它们的晶格几何形状和组成它们的孔和通道的大小方面都是可编程的，客体物种可以通过将它们拴在组装形成晶体的链上来定位(共价或通过超分子效应)。该项目将创造包含RNA和DNA的晶体，并确定这种变化对晶体对称性的影响。此外，晶体的空腔将被用来容纳RNA和DNA适配子，并使用X射线结晶学来解决它们的结构(包括目前未知的结构)。特别是小适配子很难用任何其他方式解决，因此这种方法将有助于更多这样的结构的确定。该项目的另一个目标是通过将它们连接到定点结合DNA小沟的分子上，创造出高密度的功能蛋白质(既有像GFP这样的模型系统，也有像辣根过氧化物酶这样的酶)。尽管蛋白质不会受到足够严格的束缚，不足以用结晶学来解决它们的结构，但晶格可以作为催化材料，作为蛋白质纯化的亲和支架，或者保护蛋白质不被降解。该项目的第三个关键目标是将晶体的尺寸缩小到100-200纳米(与传统的此类晶体相比，后者为数百微米)。这些纳米晶体将被用来将功能货物(如siRNA或功能蛋白质)输送到细胞内部，规则的晶格将提供极高的负载能力，例如直径约100 nm的晶体可负载约1000个分子。综上所述，这项工作将：(1)创建一套新的支架，用于确定RNA和DNA适配子结构；(2)使功能材料能够在3D空间中控制蛋白质的附着；以及(3)设计一种新的纳米颗粒，具有极高的蛋白质或siRNA负载能力，可有效地运送到细胞。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

PART 1: NON-TECHNICAL SUMMARY Nature uses self-assembly in order to organize molecules into functional materials, with biological cells being a prime example of the great potential in this approach. DNA and RNA are two of the most promising molecules for constructing tailored self-assembling systems because they encode information in a programmable way, so many independent strands can be designed that come together in a predictable manner. The molecular properties (e.g. the dimensions, geometry, and rigidity) of these nucleic acids are also well known, and they can form branched junctions that allow them to assemble in 2D and 3D space. One key goal of nucleic acid-based nanotechnology is to build 3D crystals with programmable void spaces to host various guest molecules. This project aims to show, for the first time, that molecules like RNA or proteins can be specifically positioned in these crystals, by tethering them to strands that make up the lattice. The first goal is to incorporate RNA into the crystals, both to see how it changes the assembly of the lattice, but also to solve the structure of small, unknown RNA motifs like aptamers. In the second goal, proteins will be attached to the crystals, not to solve their structure, but rather to create a dense 3D array of these molecules (e.g. for catalytic reasons, by incorporating enzymes). Finally, the third goal of this project is to create nano-crystals (which are roughly a billion-fold smaller than the crystals typically obtained), laden with either small interfering RNA (siRNA) or functional proteins. These crystals can be used to effectively deliver these cargoes into cells, given the extremely high density they can carry. Taken together, the goals in this proposal will create a new system of 3D scaffolds for solving RNA structures, attaching proteins to make catalytic materials, and more efficiently delivering important functional molecules into cells. The project will also have significant societal and educational impact by developing an online curriculum and teacher training for K-12 education and a new self-assembly online game for exploring how these systems work. This program will engage undergraduate, graduate, and underrepresented minority students to gain knowledge and pursue research in the science, technology, engineering, and math (STEM) fields, and help develop a new course for teaching nanotechnology to a broad range of students.PART 2: TECHNICAL SUMMARY The goal of this project is to use self-assembled, 3D DNA crystals as functional macromolecular scaffolds that can immobilize guest molecules, such as RNA and proteins. These DNA crystals are programmable in both their lattice geometry and the size of the pores and channels that comprise them, and guest species can be site-specifically positioned by tethering them to the strands that assemble to form the crystal (either covalently or via supramolecular effects). This project will create crystals that incorporate RNA as well as DNA, and determine the effects on crystal symmetry of this change. In addition, the cavities of the crystal will be used to host both RNA and DNA aptamers, and to solve their structure (including ones that are currently unknown) using X-ray crystallography. Small aptamers in particular are difficult to solve in any other way, and thus this method will facilitate the determination of more such structures. Another goal of the project will be to create a high density of functions proteins (both model systems like GFP, and enzymes like horseradish peroxidase), by attaching them to molecules that site-specifically bind the minor groove of DNA. Although the proteins will not be tethered rigidly enough to solve their structure using crystallography, the lattices can serve as catalytic materials, as affinity scaffolds for protein purification, or ways to protect the proteins from degradation. The third key goal of this project will be to scale down the size of the crystals to 100-200 nm in size (compared with traditional such crystals, which are hundreds of micrometers). These nano-crystals will be used to deliver functional cargoes (such as siRNA or functional proteins) to the interior of cells, and the regular lattice will provide an extremely high loading capacity, e.g. ~1,000 molecules for a crystal approximately 100 nm in diameter. Taken together, the work will: (1) create a new set of scaffolds for determining RNA and DNA aptamer structure; (2) enable functional materials with controlled attachment of proteins in 3D space; and (3) design a new category of nanoparticles with extremely high loading capacity of proteins or siRNA for effective delivery to cells.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.