UNS: Purification of plasmid DNA using enhanced ultrafiltration systems

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。实验将设计用于开发和测试具有独特孔结构的新型膜，专门设计用于延长DNA并增强整体分离。此外，已知可以选择性结合DNA的小分子将被用于通过改变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：(a)开发和测试具有锥形或梯度孔形态的膜，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，从而增强不同质粒异构体之间的分离。带锥形孔的膜将通过跟踪聚碳酸酯膜的微分蚀刻生产。通过静电纺纳米纤维或将不同孔径的膜分层成有效的复合膜，可以制备出具有梯度形态的膜。初始实验将用单个质粒同种异构体的溶液进行，使用一系列具有不同碱基对数量的质粒。实验结果将使用现有的聚合物伸长率理论模型进行分析，适当地扩展到考虑锥形孔结构。实际的质粒分离将使用在滤过模式下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分析饲料和渗透样品。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，并为开发用于治疗应用的超螺旋DNA纯化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过程和生物分子纯化的实践经验，这些技能对于支持美国生物技术和制药行业的努力至关重要。

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.