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; 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）拟议的研究的总体目标是开发增强的膜过程，以纯化超螺旋质粒DNA，这是治疗应用中感兴趣的生物活性DNA的形式。 实验将旨在开发和测试具有专门设计旨在拉长DNA并增强整体分离的独特孔结构的新型膜。 另外，已知与DNA结合的已知小分子将通过改变DNA分子的伸长柔韧性来微调分离特性。 这些结果将为开发用于治疗应用的超螺旋DNA的膜系统的开发提供基本见解和实用指南。 拟议的研究将通过：（a）使用锥形或梯度孔形态学的膜的开发和测试来解决这两个问题，这些膜可以有效地预先伸展DNA，从而最大程度地减少小毛孔入口处的DNA捕获，（b）选择性结合的使用，从而改变了伸长式的柔韧性，从而改变了峰值的柔韧性。 带有锥形（圆锥形）孔的膜将通过径移蚀刻的聚碳酸酯膜蚀刻而产生。 具有梯度形态的膜将由电纺纳米纤维或通过将具有不同孔径特性的膜分层为有效的复合膜来产生。最初的实验将使用一系列具有不同碱基对数量的质粒进行单个质粒同工型的溶液进行。 将使用可用的聚合物伸长的理论模型来分析实验结果，并适当扩展以说明锥形孔结构。实际的质粒分离将使用以隔膜模式运行的线性量表过滤模块进行，并使用馈入和渗透样品使用琼脂糖凝胶电泳和亲和力色谱法分析。 这些研究将为DNA伸长对膜孔结构和分离的影响提供基本的见解，以及开发用于纯化超螺旋DNA用于治疗应用的膜系统的实用准则。 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净化的动手经验，这些技能是支持美国生物技术和制药行业的努力的迫切需要的技能。