Self-Assembly and Dynamic Reconstruction of Expanded Biomolecular Co-Crystals

膨胀生物分子共晶的自组装和动态重建

• 批准号: 2310574
• 负责人: Christopher Snow
• 金额: $ 43.68万
• 依托单位: Colorado State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-15 至 2026-03-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2310574&HistoricalAwards=false
• 关键词: Self; Assembly; Dynamic; Reconstruction; Expanded

NON-TECHNICAL SUMMARYDynamic renewal is a key feature of natural self-healing or adaptive materials. This fundamental research program aims to create biomaterials that combine this dynamic reconstruction capacity with the extraordinarily precise organization of a crystal. Specifically, this project will use crystals composed of engineered protein, DNA, and RNA building blocks. The designed crystals will have pores large enough to permit the infiltration or controlled release of protein and nucleic acid components. Ideally, the resulting living materials will allow active addition or substitution of molecular building blocks. To unlock a variety of practical material science applications, scaffold crystals made out of biomolecules must be fortified after self-assembly. Therefore, one aspect of the project is the optimization of methods for adding bonds throughout the crystals and the quantification of the resulting stability increases. Precise control of the 3-D position of functional molecules within a scaffold crystal opens the door for materials with unprecedented performance for diverse additional applications including biosensing, catalysis, energy conversion, biomedicine, and biotechnology. For example, porous co-crystals that anchor, protect, and release functional RNAs will have applications in therapeutic RNA delivery. To partially explore these applications, the team will provide mentorship and funding for 3 years of undergraduate-led biomolecular design teams (2023, 2024, and 2025). Inspiring and training the next generation of students to innovate at the biomaterials design frontier will directly accelerate the pace of discovery, to the benefit of the scientific community and the nation.TECHNICAL SUMMARYThis research program will develop a new class of crystalline biomaterials composed of both protein and DNA building blocks, with solvent channels large enough to permit intra-crystal transport of macromolecules. The limits of co-crystal expansion modularity will be tested through assembly trials with struts composed of dsRNA, hybrid RNA:DNA, and a mixture of varying dsDNA blocks. This project explores "living" materials with actively replaceable molecular components. Confocal microscopy will be used to track the incorporation of fluorescent building blocks, as well as the site-specific capture of functional RNA added after crystal growth. Engineered co-crystals will be stabilized via chemical ligation and disulfide-based protein polymerization. Crystal stability when challenged with high temperature will be quantified via microscopy, spectroscopy, and nanopore sequencing. To demonstrate biomolecular infiltration and substitution, ligase and endonuclease domains will be diffused into the crystal interior to verify that enzymatic ligation stabilizes the crystals, but subsequent nuclease attack reverses stabilization via controlled crystal demolition. This project will then determine if endonuclease and ligase can work together to gradually excise and substitute scaffold crystal components. This project establishes a foundation for subsequent applied research. For example, porous co-crystals that anchor, protect, and release functional RNAs will have applications in therapeutic RNA delivery. Education and outreach activities associated with this project include support for a semi-autonomous undergraduate research organization, REU students, underrepresented students, first-generation college students, and high school interns. This education plan is intertwined with the research plan since a large team of mentees is critical to pursue, in parallel, the large number of proposed biomolecular crystal variants.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.

非技术总结动态更新是自然自愈或自适应材料的一个关键特征。这项基础研究计划旨在创造生物材料，结合联合收割机这种动态重建能力与晶体的非常精确的组织。具体而言，该项目将使用由工程蛋白质，DNA和RNA构建模块组成的晶体。所设计的晶体将具有足够大的孔以允许蛋白质和核酸组分的渗透或受控释放。理想情况下，所得的活性材料将允许分子构建块的活性添加或取代。为了解锁各种实际的材料科学应用，由生物分子制成的支架晶体必须在自组装后进行强化。因此，该项目的一个方面是优化在整个晶体中添加键的方法，并量化所产生的稳定性增加。精确控制功能分子在支架晶体中的三维位置为具有前所未有性能的材料打开了大门，这些材料可用于各种其他应用，包括生物传感，催化，能量转换，生物医学和生物技术。例如，锚、保护和释放功能性RNA的多孔共晶体将在治疗性RNA递送中具有应用。为了部分探索这些应用，该团队将为3年的本科生领导的生物分子设计团队（2023年，2024年和2025年）提供指导和资金。培养和培养下一代学生在生物材料设计前沿进行创新，将直接加快发现的步伐，造福科学界和国家。技术概述本研究计划将开发一类新的晶体生物材料，由蛋白质和DNA构建模块组成，具有足够大的溶剂通道，允许大分子在晶体内运输。共晶扩展模块性的极限将通过组装试验进行测试，其中支柱由dsRNA、杂交RNA：DNA和不同dsDNA块的混合物组成。该项目探索具有可主动替换的分子成分的“活”材料。共聚焦显微镜将用于跟踪荧光构建块的掺入，以及晶体生长后添加的功能RNA的位点特异性捕获。工程共晶体将通过化学连接和基于二硫化物的蛋白质聚合来稳定。当用高温挑战时的晶体稳定性将通过显微镜、光谱学和纳米孔测序来量化。为了证明生物分子的渗透和取代，连接酶和核酸内切酶结构域将扩散到晶体内部，以验证酶促连接稳定晶体，但随后的核酸酶攻击通过受控的晶体破坏逆转稳定。然后，该项目将确定核酸内切酶和连接酶是否可以一起工作，以逐渐切除和取代支架晶体成分。该项目为后续的应用研究奠定了基础。例如，锚、保护和释放功能性RNA的多孔共晶体将在治疗性RNA递送中具有应用。与该项目相关的教育和推广活动包括支持半自治的本科研究组织，REU学生，代表性不足的学生，第一代大学生和高中实习生。该教育计划与研究计划交织在一起，因为一个庞大的受指导者团队对于同时追求大量拟议的生物分子晶体变体至关重要。该奖项反映了NSF的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。