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

动态更新是自然自愈或自适应材料的一个关键特征。这个基础研究项目旨在创造生物材料，将这种动态重建能力与晶体的异常精确组织结合起来。具体来说，这个项目将使用由工程蛋白、DNA和RNA组成的晶体。所设计的晶体将具有足够大的孔，以允许渗透或控制蛋白质和核酸成分的释放。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替代。为了解锁各种实际材料科学应用，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在整个晶体中添加键的方法的优化和由此产生的稳定性增加的量化。精确控制支架晶体中功能分子的3-D位置为具有前所未有性能的材料打开了大门，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，锚定、保护和释放功能性RNA的多孔共晶体将在治疗性RNA递送中得到应用。为了部分探索这些应用，该团队将为本科生领导的生物分子设计团队（2023年、2024年和2025年）提供3年的指导和资金。激励和培训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。本研究项目将开发一类由蛋白质和DNA组成的新型晶体生物材料，其溶剂通道足够大，可以允许大分子在晶体内运输。共晶扩展模块化的限制将通过由dsRNA、杂交RNA:DNA和不同dsDNA块的混合物组成的支杆组装试验来测试。该项目探索具有主动可替换分子成分的“活”材料。共聚焦显微镜将用于跟踪荧光构建块的结合，以及晶体生长后添加的功能性RNA的特定位点捕获。工程共晶将通过化学连接和基于二硫化物的蛋白质聚合来稳定。晶体稳定性时，挑战高温将通过显微镜，光谱学和纳米孔测序定量。为了证明生物分子渗透和取代，连接酶和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控制晶体破坏逆转稳定。这个项目将确定是否核酸内切酶和连接酶可以一起逐渐去除和替代支架晶体成分。本课题为后续的应用研究奠定了基础。例如，锚定、保护和释放功能性RNA的多孔共晶体将在治疗性RNA递送中得到应用。与此项目相关的教育和推广活动包括支持半自治的本科生研究组织、REU学生、代表性不足的学生、第一代大学生和高中实习生。这个教育计划与研究计划交织在一起，因为一个庞大的学员团队对于并行地追求大量提出的生物分子晶体变体至关重要。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值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.