PROJECT SUMMARY/ABSTRACT: A number of candidate therapies such as CRISPR-Cas9 and gene silencing require the efficient delivery of functional nucleic acids to the cell cytosol and nucleus. Unfortunately, such therapies currently lack proper delivery mechanisms, precluding their widespread applicability. Self- assembled deoxyribonucleic acid (DNA) nanoparticles have shown potential as minimally cytotoxic therapeutic carriers in cancerous and other in vitro and in vivo models. While evidence suggests that DNA nanoparticles-based drug carriers can be taken up by mammalian cells via endocytosis, it is unknown how these DNA nanoparticles can overcome the fate of endocytosis-triggered degradation to reach the cytosol and, once there, can controllably maintain stability. With the enabling science explaining their behavior and mechanisms of controlling their stability in the cell cytosol it will be possible to make bold advances in engineering therapeutic delivery systems. To that end, the proposed work has two overarching scientific payoffs. Payoff 1, induce endosomal escape and enhanced cytosolic accessibility of DNA nanoparticles by the integration of calcium in their assembly process. Payoff 2, identify the rate of breakdown and mechanisms of stabilization of DNA nanoparticles in different types of cell cytosols. Innovative technologies will be the foci of the PI's training program and will be implemented to achieve the project goals, namely, multi-step Förster resonance energy transfer spectroscopy for high-resolution tracking of DNA nanoparticle inside the cell and in vitro cell microinjections enabling study of these nanoparticles directly in the cytosolic environment. First, a DNA origami based nanotube will be tested for structural stability in calcium-supplemented buffer. Thereafter, the nanotube will be used as a carrier for the delivery of functional RNA molecules to representative fluorescent protein-expressing cells and checked for its cytosolic reachability and efficacy in protein regulation after undergoing endocytosis. Second, small (20 nm) DNA nanoparticles with branched architecture and non-canonical nucleic acids will be embedded with multi-step FRET reporters for measuring structural integrity. These DNA nanoparticles will be microinjected into live cells cytosolic region and their breakage be determined. Last, the cytosolic stability of these DNA nanoparticles will be correlated with different types of mammalian cells with known cytosolic variability (tumor, immune, and other cell types) in order to map the role of structurally diverse DNA nanoparticles in targeting cells with different physiologies. The PI will also receive training in rigorous analysis of in vitro research, lab management, and the prolific grant writing process, which will facilitate their transition to an independent research program. Outcomes of this project will pave the way towards developing more bio-compatible delivery systems, specifically for functional nucleic acid therapeutic agents that are vital in the cell cytosol.

PROJECT NARRATIVE Synthetic DNA-based nanoparticles possess the potential to develop advanced delivery tools for targeted therapies, but their behavior in the cytosolic environment remains poorly understood. Herein, we seek to address critical issues about the accessibility and breakdown of these nanoparticles within the mammalian cell cytosol and identify mechanisms of stabilization. Outcomes of the proposed project will enable the realization of more- biocompatible delivery systems.