Title: Development of Functionally Orthogonal DNA Origami Scaffolds for Digital Nucleic Acid Memory

Program: Biology MS

Committee Chair: Eric Hayden

Committee: Eric Hayden, Juliette Tinker, Jeunghoon Lee

Abstract: DNA nanotechnology exploits the programmable base-pairing properties of DNA to assemble nanoscale structures with precise spatial organization. Among these approaches, DNA origami enables the folding of a long single-stranded DNA scaffold into user-defined shapes through hundreds of short staple strands. This design strategy has enabled applications across nanotechnology, including molecular sensing, nanoscale fabrication, and emerging DNA-based information storage systems. One such system, digital nucleic acid memory (dNAM), encodes information through spatial patterns on DNA origami structures that can be read using super-resolution microscopy. However, most DNA origami designs rely on scaffold sequences derived from the M13 bacteriophage genome, limiting scaffold diversity and constraining systems that require multiple independently folding DNA origami structures. This work explores the development of orthogonal DNA origami scaffolds to expand scaffold diversity and support scalable implementations of DNA origami-based technologies. Specifically, we evaluate evaluate custom, randomly generated, DNA scaffolds generated using ScaffoldSmith and investigate whether these sequences can function as orthogonal scaffolds during DNA origami assembly. A set of three ~11 kb scaffolds with low sequence similarity were synthesized and used to assemble DNA origami structures compatible with dNAM architectures. Folding experiments performed in the presence of competing staple sets demonstrated robust origami assembly without statistically significant loss of yield or structural quality, indicating functional orthogonality among the tested scaffolds. These results demonstrate that computationally generated scaffolds can support reliable DNA origami folding while minimizing unintended interactions between structures. Overall, this work establishes an experimental foundation for expanding libraries of orthogonal DNA origami scaffolds. Such scaffold libraries may enable parallel synthesis of multiple DNA origami structures and support the development of scalable DNA nanotechnology systems, including future implementations of dNAM.