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

脱氧核糖核酸（DNA）表现出超越其作为遗传信息的核心作用的独特性质，使其成为制造传感器，逻辑门，计算机和复杂的折纸结构的多功能工具。这一集体应用领域被称为DNA纳米技术，依赖于对DNA序列的精确控制来实现多种功能，对人类健康和工业具有重大意义。DNA纳米技术面临的一个重大挑战是需要扩大DNA生产的非典型应用，而不会成为成本高昂，环境有害或过于繁琐。为了应对这一挑战，该项目利用制药公司在COVID-19大流行期间用于生产核酸疫苗的生物反应器来扩大DNA生产。通过使用微生物，类似于酵母将小麦转化为啤酒，这种具有成本效益和可扩展的方法有可能生产出比当前方法产量高数十万倍的DNA。在这种规模下，DNA序列之外的独特性质可以用来创造具有新特性的材料。与几乎任何其他分子不同，DNA的形状可以控制并编织成奇异的形式，以赋予其增强的特性，就像编织图案可以控制织物的强度一样。此外，DNA与蛋白质和其他分子的相互作用为精确操作提供了新的机会，从而可以创造可调材料。这项研究，其中包括生物反应器的跨学科应用，提供了围绕DNA纳米技术的两个教育机会。“科学混搭”计划允许高中生融合两门科学，如化学和生物学，同时通过现场演示展示令人兴奋的结果。此外，生物反应器靴子营提供给本科生和研究生，装备他们的必要技能，操作生物反应器，同时促进与科学家从龙沙的朴茨茅斯NH设施，在工业生物反应器的当地专家互动。总的来说，这项研究的目的是提升DNA纳米技术到一个更广泛的规模，并产生新的材料赋予创新和可控的结构-properties.Technical Summary：这项研究的目的是利用廉价的，可扩展的，和环境友好的生产双链DNA（dsDNA）的生物反应器，以产生DNA水凝胶。尽管DNA水凝胶的例子很多，但与成本、可持续性和批量制备相关的挑战阻碍了这些材料在许多最终用途应用中的转化。支持这一研究进展的假设是，获得克级数量的双链DNA（dsDNA）将提供新的途径来获得利用dsDNA独特的聚合物性质的散装材料，并获得对其结构-性质关系的前所未有的控制和洞察。本研究的主要目标是推进对DNA水凝胶的基本理解，并建立决定其结构-性质关系的设计原则。新的创新方法的发展使得在学术实验室环境中能够以克级规模生产DNA双链体。然后，这些双链体作为结构单元，通过共价、超分子和酶促方法产生大量dsDNA水凝胶。通过重新利用用于研究基因表达和DNA拓扑结构的策略，目的是获得对水凝胶网络拓扑结构的前所未有的控制，并阐明其基本性质。最初的研究重点是扩大从生物反应器中纯化和衍生质粒DNA（pDNA），促进水凝胶材料的成本效益和高效生产。随后，这些材料在物理和共价连接的水凝胶的结构特征和机械和化学性质之间的连接建立。最终，教育部分通过提供全面的示范和培训，重点是生物反应器和DNA水凝胶的有效利用，利用研究的跨学科性质。预期的创新包括：（一）发展负担得起的、方便的、和可持续的方法来获得dsDNA水凝胶（II）通过新的交联策略实现的独特的整体性质的系统研究和（III）聚合物网络拓扑结构和缠结与整体力学性能的量化和相关性。该奖项反映了NSF的法定使命，并通过使用基金会的智力价值进行评估，被认为值得支持和更广泛的影响审查标准。

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 plasmidDNA (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.