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

Colorado State University, Fort Collins CO

Investigators

Abstract

NON-TECHNICAL SUMMARY Dynamic 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 SUMMARY This 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.