POROUS PROTEIN-DNA CO-CRYSTALS FOR NUCLEIC ACID CAPTURE, STORAGE, AND RELEASE

Abstract

Porous biomolecular crystals provide an emerging platform for nanoscale organization, molecular capture, and controlled release, but a system that combines modularity, tunability, and high-resolution structural precision has yet to be elucidated. Biomolecular nanoparticles made of pure protein and pure DNA have been explored for use in biomedical, structural, and nanotechnology applications. By combining the structural rigidity of protein crystals and the modularity of DNA crystals, we have developed a class of biomolecular co-crystals that fills gaps currently present in the biomolecular materials field. We are interested in how protein-DNA co-crystals can be used as scaffolds, hosting guest molecules by soaking them in after the protein-DNA co-crystals have formed. Unique to our platform among crystalline biomaterials capable of spatial organization of guest molecules is the ability to diffract X-rays at a resolution sub 3 Angstroms. This dissertation presents a comprehensive exploration of these high-resolution protein–DNA co‑crystals as programmable crystalline nanoparticles engineered for selective nucleic acid sequestration, stabilization, and triggerable release. We review the landscape of biomolecular crystalline nanoparticles in biomedical applications, highlighting how their periodicity and defined pore environments enable drug loading, biosensing, and molecular templating. To enable our protein-DNA co-crystal to persist in the stringent environments necessary to be of service in these fields, we describe efforts to enhance the stability of protein–DNA co‑crystals by optimizing crosslinking at DNA–DNA junctions using both chemical crosslinkers and enzymatic ligation strategies. These approaches significantly increase crystal robustness while preserving porosity and modular programmability. To demonstrate the utility of having programmable DNA elements that take advantage of scaffold protein rigidity, we next show the chemical and structural diversity of the system by incorporating non‑canonical nucleobases, alternative backbone chemistries, and engineered secondary structures. We then leverage these design principles to show nucleic‑acid capture from the environment, storage within the crystal pores, and specific release mechanisms using toehold‑mediated strand displacement, showcasing highly specific, trigger‑dependent unloading of target cargo from protein-DNA co-crystals. For practical use of our platform in biomedical applications, we adapt microcrystallization methods to enable scalable production and consistency in nanoparticle batches. To exemplify quantifiable guest molecule loading within our co-crystals, we present a novel cuvette-based uptake fluorescence assay. Finally, we outline future directions for this work in structural, biomedical, and nanotechnology fields. Together, this work establishes protein–DNA co‑crystals as a modular, engineerable material system capable of selective nucleic acid capture, stable storage, and programmable release. This opens new avenues for responsive biomaterials, biosensing, and therapeutic delivery.