Collaborative Research: Designing Multivalent Ligands for Plasmid DNA Purification

1067840/1066998 Rege/Jayaraman该合作提案的总体研究目标是对用于质粒DNA纯化的多价配体的分子工程和基本机理的理解。基因治疗已成为一种有吸引力的治疗策略，以遗传畸变为特征的疾病。目前，超过四分之一的基因治疗临床试验使用质粒DNA（pDNA）。对pDNA需求的增加将需要有效的纯化方法来成功地将治疗性pDNA从实验室工作台转移到患者床边。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色谱方法，但其特征在于低容量、选择性和产率，这主要是由于pDNA独特的物理化学性质。对pDNA具有高容量和/或选择性的新配体的发现将导致高效且成本有效的分离方法。假设在分子支架上掺入多个拷贝的假亲和力DNA结合分子将产生对pDNA具有高结合亲和力的多价配体（ML）。分子动力学（MD）模拟将被用来消除弱结合的ML和调查基本的物理化学现象的配体-DNA结合。将合成一个由MD模拟指导的ML小型库。将以平行方式评价ML与质粒DNA的结合，结合数据的平衡热力学建模将阐明ML与pDNA结合中多价性和协同性的作用。将高亲和力ML与有机和无机膜结合，以从细胞培养液中纯化质粒DNA。这种膜色谱方法是基于ML导致的高结合能力和膜大孔（400 nm）中的低转运阻力的组合。因此，所提出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的基本见解，以及使用膜色谱法进行pDNA纯化。这项研究将对未来的生物加工产生重大影响，从而降低用于基因治疗和DNA疫苗的质粒DNA的成本，这对社会有巨大的好处。拟议的工作将最终在分子相互作用，多价性和合作性的配体发现，这可能是有用的，涉及生物分子的纯化的其他应用程序的作用的基本理解。在PI Rege和PI Jayaraman的指导下，参与该项目的两名研究生将接受模拟和实验技术方面的全面培训，培训方式为a）通过Skype每月举行一次项目进展会议，B）在项目期间进行一次为期6个月的学生交流。并分别在亚利桑那州立大学和科罗拉多大学培训本科生。虽然PI Rege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，PI Jayaraman通过本科生研究机会计划（UROP）支持本科生在科罗拉多大学（CU）。作为拟议的外联活动的一部分，PI Rege最近与梅萨的梅萨公共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PI将整合研究生和本科生在实验室演示的参与，在推广活动，以高中学生。参加外展活动的研究生和本科生将受益于外展计划中提出的教学和指导活动。预计该推广方案将大大有助于凤凰城大都会和丹佛地区的高中教育，并将激励女学生和来自代表性不足人口的学生继续接受科学和工程教育

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