UNS: Purification of plasmid DNA using enhanced ultrafiltration systems

1505592（Zydney）所提出的研究的总体目标是开发用于纯化超螺旋质粒DNA的增强的膜过程，超螺旋质粒DNA是治疗应用中感兴趣的DNA的生物活性形式。 实验将被设计为开发和测试具有独特孔结构的新型膜，这些孔结构专门设计用于延长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分子的延伸柔性来微调分离特性。 这些结果将为开发用于治疗应用的超螺旋DNA纯化的膜系统提供基本的见解和实践指南。 拟议的研究将解决这两个问题，通过：（a）开发和测试的膜锥形或梯度孔形态，可以有效地预拉伸的DNA，从而最大限度地减少DNA捕获在入口处的小孔，和（B）使用选择性结合配体，以改变延伸的灵活性，从而提高不同的质粒异构体之间的分离。 将通过跟踪聚碳酸酯膜的差异蚀刻来生产具有锥形（圆锥形）孔的膜。 具有梯度形态的膜将通过电纺纳米纤维或通过将具有不同孔径特征的膜分层成有效的复合膜来产生。将使用具有不同碱基对数量的一系列质粒，使用单个质粒同种型的溶液进行初始实验。 实验结果将使用聚合物伸长率的可用理论模型进行分析，适当扩展以考虑锥形孔结构。将使用以渗滤模式运行的线性可扩展过滤模块进行实际质粒分离，并使用琼脂糖凝胶电泳和亲和色谱法分析进料和渗透液样品。 这些研究将提供基本的见解，DNA延伸对膜孔结构和分离的影响，以及为开发用于治疗应用的超螺旋DNA纯化的膜系统的实际指导方针。 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努力所急需的。

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.