Publication Date

12-2022

Date of Final Oral Examination (Defense)

10-20-2022

Type of Culminating Activity

Dissertation

Degree Title

Doctor of Philosophy in Biomolecular Sciences

Department

Biology

Supervisory Committee Chair

Eric J. Hayden, Ph.D.

Supervisory Committee Member

Daniel Fologea, Ph.D.

Supervisory Committee Member

Lisa Warner, Ph.D.

Abstract

Recently, DNA nanotechnology has emerged as a promising and rapidly expanding method to utilize nucleic acids as a nanoscale building material. DNA origami is a major structural application of DNA nanotechnology, using DNA to construct two- and three-dimensional shapes. These structures have been employed for a variety of uses including DNA data storage. DNA is a promising material to address the impending shortage of silicon-based storage as data demands increase. There are many sequence-based methods of data storage, but digit Nucleic Acid Memory (dNAM) uses DNA origami as a breadboard and is read by super-resolution microscopy instead. dNAM uses DNA origami to spatially position DNA probe sequences in a matrix arrangement that can be read by DNA-PAINT. The prototype used 15 different origami structures to successfully encode and read “Data is in our DNA!\n”.

The dNAM prototype showed the feasibility of using DNA origami as a breadboard, however, the origami’s size limits data capacity and reading efficiency. In chapter 2, we engineered a larger DNA origami rectangle for dNAM. First, we designed a larger node, with an 8x10 matrix of potential data points, a 67% increase from the dNAM prototype. To construct this larger structure, we designed, cloned, produced, and tested a large, custom ssDNA scaffold. With this scaffold, we successfully folded larger origami as confirmed by AFM, and showed the correct positioning of DNA data point probes by DNA-PAINT. This larger structure enabled a 67% increase in the number of data points per origami, which allows for an 80% increase in the number of bits/node when encoding data. This larger node supports the scaling of dNAM, and will allow for more efficient production and reading.

To take advantage of recent advances in array-based oligonucleotide synthesis, in chapter three we explore the use of pooled staples for dNAM. First, we tested the performance of pooled staples compared to individually synthesized staples using the original dNAM node with the M13mp18 scaffold. We showed that both sets of staples performed equally well in terms of folding origami, and arranging the matrix of data points. Next, we tested the formation of multiple origami structures using orthogonal scaffolds in the context of mixed pools of oligos. We compared the ability of two different scaffolds to fold into the appropriate origami with individual and mixed sets of staple strands. We showed that origami could be folded successfully with either one scaffold and both sets of staples (“random access”) or both scaffolds and both sets of staples (“one-pot synthesis”). Finally, we designed multiple scaffolds that use orthogonal sets of staple strands and analyzed their orthogonality. Together, these results move dNAM towards taking advantage of pooled oligos, which will enhance scalability and efficiency. All moving dNAM towards real world applications.

DOI

https://doi.org/10.18122/td.2025.boisestate

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