DNA Canvas: Towards affordable and scalable enzymatic fabrication of DNA nanoarrays

Abstract

In biological systems, DNA serves as a carrier of hereditary information, facilitated by predictable and programmable base-pairing rules. The field of DNA nanotechnology takes the DNA molecule out of its original context and using the same set of rules to construct complex structures and molecular machines at the nanoscale regime. At the nanoscale, precise organization of biological and non-biological materials in 2D or 3D space holds great promise for a vast range of applications in areas such as biophysics, point-of-care diagnostics, biomolecule structure determination, drug delivery, and more. Nucleic acid scaffolds, especially DNA origami, have emerged as a promising approach, by enabling <10nm assembly of nanomaterials such as gold particles, carbon nanotubes, and quantum dots. Two-dimensional DNA nanostructures with a plurality of uniquely-addressable linkage sites ("nanopixels") are known as DNA nanoarrays.

Expanding the size of DNA nanoarrays is desired for a variety of applications, from whole genomic sequencing at a fraction of the cost to sustainable digital information storage. Yet, due to the stochastic nature of self-assembly, DNA origami-based approaches suffer from an inherent scale limit. Top-down fabrication techniques enable nanometric precise patterning, yet single-molecule placement remains a daunting challenge. Currently, no method enables independent nanoscale manipulation of more than 10K diverse single-molecules.

In this thesis, we propose a new kind of DNA nanotechnology: DNA Canvas. We lay a theoretical and experimental foundation for the development of a low-cost DNA nanoarray. First, we present a computational model as feasibility proof, as well as statistical analysis of the experimental design space, aiming to minimize the cost per nanopixel. We demonstrate a novel fabrication process that fuses microfabrication by photolithography with enzymatic reactions to surpass the scale limit in previous approaches. Last, we chart a path towards full implementation of >1M DNA nanoarrays and enumerate potential applications to be disrupted by the introduction of such technology.