UNS: Purification of plasmid DNA using enhanced ultrafiltration systems

UNS：使用增强型超滤系统纯化质粒 DNA

• 批准号: 1505592
• 负责人: Andrew Zydney
• 金额: $ 30.85万
• 依托单位: Pennsylvania State Univ University Park
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2019-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1505592&HistoricalAwards=false
• 关键词: UNS; Purification; plasmid; DNA; using

1505592 (Zydney)The overall objective of the proposed research is to develop enhanced membrane processes for the purification of supercoiled plasmid DNA, the biologically active form of DNA of interest in therapeutic applications. Experiments will be designed to develop and test novel membranes with unique pore structures specifically designed to elongate the DNA and enhance the overall separation. In addition, small molecules that are known to selectively bind to DNA will be used to fine tune the separation characteristics by altering the elongational flexibility of the DNA molecules. These results will provide fundamental insights and practical guidelines for the development of membrane systems for purification of supercoiled DNA for therapeutic applications. The proposed research will address both of these issues through: (a) the development and testing of membranes with tapered or gradient pore morphologies that can effectively pre-stretch the DNA and thus minimize DNA trapping at the entrance to the small pores, and (b) the use of selective binding ligands to alter the elongation flexibility and thus enhance the separation between different plasmid isoforms. Membranes with tapered (conical) pores will be produced by differential etching of tracked polycarbonate membranes. Membranes with gradient morphologies will be produced by electrospun nanofibers or by layering membranes with different pore size characteristics into an effective composite membrane. Initial experiments will be performed with solutions of the individual plasmid isoforms, using a series of plasmids with different numbers of base pairs. Experimental results will be analyzed using available theoretical models for polymer elongation, appropriately extended to account for the tapered pore structure. Actual plasmid separations will be performed using linearly-scalable filtration modules operated in a diafiltration mode, with feed and permeate samples analyzed using both agarose gel electrophoresis and affinity chromatography. These studies will provide fundamental insights into the effects of DNA elongation on membrane pore structure and separation as well as practical guidelines for the development of membrane systems for purification of supercoiled DNA for therapeutic applications. In addition, the proposed research program will provide graduate and undergraduate students with hands-on experience dealing with membrane processes and biomolecule purification, skills that are critically needed to support the efforts of the U.S. biotechnology and pharmaceutical industries.

1505592 (Zydney) 拟议研究的总体目标是开发用于纯化超螺旋质粒 DNA 的增强膜工艺，超螺旋质粒 DNA 是治疗应用中感兴趣的 DNA 的生物活性形式。 实验将旨在开发和测试具有独特孔结构的新型膜，这些孔结构专门用于延长 DNA 并增强整体分离。 此外，已知选择性结合 DNA 的小分子将用于通过改变 DNA 分子的伸长灵活性来微调分离特性。 这些结果将为开发用于纯化治疗应用的超螺旋 DNA 的膜系统提供基本见解和实用指南。 拟议的研究将通过以下方式解决这两个问题：(a) 开发和测试具有锥形或梯度孔形态的膜，该膜可以有效地预拉伸 DNA，从而最大限度地减少小孔入口处的 DNA 捕获，以及 (b) ）使用选择性结合配体来改变延伸灵活性，从而增强不同质粒亚型之间的分离。 具有锥形孔的膜将通过跟踪聚碳酸酯膜的差异蚀刻来生产。 具有梯度形态的膜将通过静电纺丝纳米纤维或通过将具有不同孔径特征的膜分层形成有效的复合膜来生产。初始实验将使用各个质粒亚型的溶液进行，使用一系列具有不同数量碱基对的质粒。 将使用可用的聚合物伸长理论模型来分析实验结果，并适当扩展以考虑锥形孔结构。实际的质粒分离将使用以渗滤模式操作的线性可扩展过滤模块进行，并使用琼脂糖凝胶电泳和亲和层析对进料和渗透物样品进行分析。 这些研究将为 DNA 延伸对膜孔结构和分离的影响提供基本见解，并为开发用于纯化治疗应用的超螺旋 DNA 的膜系统提供实用指南。 此外，拟议的研究计划将为研究生和本科生提供处理膜工艺和生物分子纯化的实践经验，这些技能是支持美国生物技术和制药行业所急需的技能。