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

非技术总结动态更新是自然自我修复或适应性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，将这种动态重建能力与极其精确的晶体组织结合在一起。具体地说，该项目将使用由工程蛋白质、DNA和RNA构建块组成的晶体。设计的晶体将具有足够大的孔，以允许蛋白质和核酸成分的渗透或受控释放。理想情况下，由此产生的生物材料将允许分子构建块的活性添加或替代。为了开启各种材料科学的实际应用，由生物分子制成的支架晶体必须在自组装后进行加固。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，并对由此产生的稳定性增加进行量化。对功能分子在支架晶体中三维位置的精确控制为具有前所未有的性能的材料打开了大门，这些材料可用于各种额外的应用，包括生物传感、催化、能源转换、生物医学和生物技术。例如，锚定、保护和释放功能RNA的多孔共晶将在治疗性RNA输送中应用。为了部分探索这些应用，该团队将为本科生领导的三年生物分子设计团队(2023年、2024年和2025年)提供指导和资金。激励和培养下一代学生在生物材料设计前沿创新，将直接加快发现的步伐，造福科学界和国家。技术总结这项研究计划将开发一种由蛋白质和DNA构建块组成的新型晶体生物材料，具有足够大的溶剂通道，允许大分子在晶体内传输。共晶膨胀模数的极限将通过由dsRNA、杂交RNA：DNA和不同dsDNA块的混合物组成的支柱的组装试验来测试。该项目探索具有活性可替代分子成分的“活”材料。共聚焦显微镜将被用来跟踪荧光构建块的掺入，以及晶体生长后添加的功能RNA的特定部位捕获。工程共晶将通过化学连接和基于二硫键的蛋白质聚合来稳定。晶体在高温下的稳定性将通过显微镜、光谱分析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共同作用，逐步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，锚定、保护和释放功能RNA的多孔共晶将在治疗性RNA输送中应用。与该项目相关的教育和外展活动包括支持半自主的本科研究组织、REU学生、代表性不足的学生、第一代大学生和高中实习生。这一教育计划与研究计划交织在一起，因为一大群受训者对于同时研究大量拟议的生物分子晶体变体至关重要。这一奖项反映了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。

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.