Collaborative Research: Designing Multivalent Ligands for Plasmid DNA Purification

1067840/1066998 Rege/Jayaraman这项合作计划的总体研究目标是对用于提纯质粒DNA的多价配体的分子工程和基本机理的了解。基因治疗已经成为治疗以遗传异常为特征的疾病的一种有吸引力的治疗策略。目前正在进行的基因治疗临床试验中，超过四分之一使用了质粒DNA(PDNA)。对PDNA日益增长的需求将需要有效的纯化方法，以成功地将治疗性PDNA从实验室转移到患者床边。目前使用的质粒DNA纯化策略主要依赖于最初为蛋白质开发的配体和层析方法，但由于PDNA独特的物理化学性质，其容量、选择性和产率都较低。对PDNA具有高容量和/或选择性的新配体的发现将导致高效和经济的分离过程。假设在分子支架上掺入多个拷贝的伪亲和力DNA结合分子将导致与PDNA具有高亲和力的多价配体(MLS)。分子动力学(MD)模拟将被用来消除弱结合的MLS，并研究潜在的配体-DNA结合的基本物理化学现象。在MD模拟的指导下，将合成一个小型的MLS库。将以平行的方式评估MLS与质粒DNA的结合，结合数据的平衡热力学模拟将导致阐明多价性和协作性在ML与PDNA结合中的作用。将高亲和力的MLS偶联到有机膜和无机膜上，从细胞培养液中纯化出质粒DNA。这种膜层析方法是基于MLS的高结合能力和膜大孔(400 Nm)的低传输阻力的组合。因此，这项拟议的研究将导致发现用于PDNA纯化的新型多价配体，对这些配体的结合活性有基本的了解，并使用膜层析来纯化PDNA。这项拟议的研究将对未来的生物处理产生重大影响，从而降低用于基因治疗和DNA疫苗的质粒DNA的成本，这对社会具有巨大的好处。这项拟议的工作将从根本上理解分子相互作用、多价性和协作性在配体发现中的作用，这可能有助于其他涉及生物分子纯化的应用。参与这一拟议项目的两名研究生将在PiRege和PiJayaraman的指导下接受全面的模拟和实验技术培训，通过a)通过Skype每月召开项目进度会议，以及b)在项目过程中通过为期6个月的学生交流，此外，两家PI分别致力于整合亚利桑那州立大学和科罗拉多州本科生的研究、教育和培训。皮雷格是亚利桑那州立大学富尔顿本科生研究计划(FURI)的积极参与者，皮亚拉曼通过科罗拉多大学(CU)本科生研究机会计划(UROP)为本科生提供支持。作为拟议的外联活动的一部分，皮雷格最近与梅萨的梅萨公立学校生物技术学院建立了合作关系。Pi Jayaraman是科罗拉多大学工程学院多样性行动委员会的成员，并将继续参加他们的外联活动。这两个绩效指标将把研究生和本科生在面向高中生的外展活动中参与实验室演示结合在一起。参加外展活动的毕业生和本科生将从外展计划中提出的教学和指导活动中受益。预计外展方案将大大促进凤凰城和丹佛地区的高中教育，并将激励女学生和那些来自代表人数不足的人口的学生继续接受科学和工程方面的进一步教育。

1067840/1066998Rege/JayaramanThe overall research objective of this collaborative proposal is the molecular engineering and fundamental mechanistic understanding of multivalent ligands for plasmid DNA purification. Gene therapy has emerged as an attractive therapeutic strategy for diseases characterized by genetic aberrations. Over one-quarter of gene therapy clinical trials currently underway employ plasmid DNA (pDNA). Increasing demand for pDNA will require efficient purification methods to successfully transition therapeutic pDNA from the laboratory bench to the patient bedside. Currently used plasmid DNA purification strategies largely rely on ligands and chromatographic methods originally developed for proteins but are characterized by low capacities, selectivities, and yields mainly due to the unique physicochemical properties of pDNA. The discovery of new ligands with high capacities and/or selectivites for pDNA will lead to efficient and cost-effective separation processes. It is hypothesized that incorporation of multiple copies of pseudo-affinity DNA binding molecules on molecular scaffolds will lead multivalent ligands (MLs) with high binding affinities to pDNA. Molecular dynamics (MD) simulations will be employed to eliminate weakly binding MLs and investigate fundamental physicochemical phenomena underlying ligand-DNA binding. A small library of MLs, guided by MD simulations, will be synthesized. MLs will be evaluated for binding plasmid DNA in a parallel fashion, and equilibrium thermodynamic modeling of binding data will lead to elucidation of the roles of multivalency and co-operativity in ML binding to pDNA. High-affinity MLs will be conjugated to organic and inorganic membranes in order to purify plasmid DNA from cell culture broths. This membrane chromatography approach is based on the combination of high binding capacities due to MLs and low transport resistances in large pores ( 400 nm) of the membranes. The proposed research will therefore lead to the discovery of novel multivalent ligands for pDNA purification, fundamental insights into the binding activity of these ligands, and the use of membrane chromatography for pDNA purification. The proposed research will significantly impact future bioprocessing, leading to lowering the cost of plasmid DNA for gene therapy and DNA vaccines, which is of tremendous benefit to society. The proposed work will culminate in a fundamental understanding of the role of molecular interactions, multivalency and co-operativity for ligand discovery which can be useful for other applications involving purification of biomolecules. The two graduate students working on this proposed project will receive a well-rounded training both in simulation and experimental techniques under the guidance of PI Rege and PI Jayaraman through a) monthly project progress meetings through Skype and b) through a 6-month student exchange once during the course of the project, Additionally both PIs are committed to integrating research, education, and training of undergraduate students at ASU and Colorado, respectively. While PI Rege is an active participant in the Fulton Undergraduate Research Initiative (FURI) program at ASU, PI Jayaraman has supported undergraduates through the Undergraduate Research Opportunities Program (UROP) at University of Colorado (CU). As part of proposed outreach activity, PI Rege has recently established a collaboration with the Mesa Pubic Schools Biotechnology Academy in Mesa. PI Jayaraman is a member of the Diversity Action Committee in the College of Engineering at University of Colorado and will continue to participate in their outreach activities. Both PIs will integrate the participation of graduate and undergraduate students in laboratory demonstrations during outreach activities to the high-school students. Graduate and undergraduate in the outreach activity will benefit from the teaching and mentoring activity proposed in the outreach program. It is anticipated that the outreach program will contribute significantly to high school education in the Phoenix Metropolitan and Denver areas and will motivate women students and those from underrepresented populations to pursue further education in sciences and engineering