CAREER: Sustainable DNA Hydrogel Production via Bioreactor-Derived Plasmid DNA

Non-Technical Description:

Deoxyribonucleic acid, or DNA, exhibits unique properties extending beyond its central role as genetic information, making it a versatile tool for creating sensors, logic gates, computers, and intricate origami-like structures. This collective field of applications, known as DNA nanotechnology, relies on the precise control of DNA sequences to achieve diverse functions, holding significant implications for human health and industry.

A substantial challenge facing DNA nanotechnology is the need to scale up DNA production for atypical applications without becoming cost-prohibitive, environmentally harmful, or overly cumbersome. To address this challenge, this project leverages bioreactors, used by pharmaceutical companies for producing nucleic acid vaccines during the COVID-19 pandemic, to scale up DNA production.

By employing microorganisms, akin to how yeast transforms wheat into beer, this cost-effective and scalable approach has the potential to produce DNA in quantities that are hundreds of thousands of times greater than current methods yield. At this scale, the unique properties of DNA beyond its sequence can be harnessed to create materials with novel characteristics.

Unlike almost any other molecule, DNA's shape can be controlled and woven into exotic forms to give it enhanced properties, like how a weave pattern can control the strength of a fabric. Furthermore, DNA's interactions with proteins and other molecules offer new opportunities for precise manipulation, allowing the creation of tunable materials. This research, which encompasses the interdisciplinary application of bioreactors, provides two educational opportunities centered around DNA nanotechnology.

The 'Science Mash-up' program allows high schoolers to fuse two sciences, such as chemistry and biology, while showcasing the exciting results through live demonstrations. Additionally, a bioreactor boot camp is offered to both undergraduate and graduate students, equipping them with the necessary skills to operate a bioreactor while facilitating interactions with scientists from Lonza's Portsmouth NH facility, local experts in industrial bioreactors.

Overall, this research aims to elevate DNA nanotechnology to a broader scale and generate new materials endowed with innovative and controllable structure-properties. Technical Summary:

The objective of this research is to leverage inexpensive, scalable, and environmentally benign production of double-stranded DNA (dsDNA) from bioreactors to generate DNA hydrogels. Despite numerous examples of DNA hydrogels, challenges related to cost, sustainability, and bulk preparation hinder the translation of these materials in many end-use applications.

The hypothesis underpinning this research advance is that access to gram-scale quantities of double-stranded DNA (dsDNA) will provide new paths to obtain bulk materials that utilize dsDNA’s unique polymeric properties and gain unprecedented control and insight into their structure-property relationships. The primary goal of this research is to advance the fundamental understanding of DNA-hydrogels and establish design principles that dictate their structure-property relationships.

The development of new innovative methodologies enables gram-scale production of DNA synthons within academic laboratory settings. These synthons then serve as building blocks to create bulk dsDNA hydrogels through covalent, supramolecular, and enzymatic methods. By repurposing strategies used to study gene expression and DNA topology, the aim is to gain unprecedented control over hydrogel network topology and elucidate their fundamental properties. The initial research focuses on expanding the purification and derivation of plasmid

DNA (pDNA) from bioreactors, facilitating the cost-effective and efficient production of hydrogel materials. Subsequently, connections between the structural characteristics and the mechanical and chemical properties of these materials in both physical and covalently linked hydrogels are established. Ultimately, the educational component capitalizes on the interdisciplinary nature of the research by offering comprehensive demonstrations and training focused on the effective utilization of bioreactors and DNA hydrogels.

The expected innovations include: (I) The development of affordable, facile, and sustainable methods to access dsDNA hydrogels (II) The systematic investigation of unique bulk properties achieved through new cross-linking strategies and (III) The quantification and correlation of polymer network topology and entanglement with bulk mechanical properties.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.