The 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
PI Arthi Jayaraman, University of Colorado at Boulder Intellectual Merit: Plasmid DNA is an attractive therapeutic molecule for gene therapy, DNA vaccines, and for several in vitro applications. As a result, there is an immense need for novel effective methods to separate pure plasmid DNA from bacterial cell culture broths. In this project, our goal has been to design polyamine based ligands that when functionalized onto membranes will bind to the plasmid DNA strongly and lead to plasmid DNA separation. The systems of interest included multivalent ligands consisting of polyamines - spermine and dilysines- grafted to aminoglycoside core – neamine, kanamycin and neomycin. The hypothesis was that grafted of the polyamines to the aminoglycoside core will improve its binding affinity towards plasmid DNA. The experimental component of the proposed work was conducted in Dr. Rege’s lab at Arizona State University and the computational aspect of the proposed research was conducted in Dr. Jayaraman’s lab at UColorado. The PIs maintained a feedback loop where the computational results from PI Jayaraman’s lab were used to first interrogate mechanisms behind some of the experimental results previously obtained by PI Rege (e.g. DNA binding affinities of neamine tetraspermine and kanamycin tetraspermine), and after satisfactorily validating the computational approach, Jayaraman’s team has made predictions of new multivalent ligands (e.g. neomycin hexa-spermine and lysine-based ligands) and their binding affinity to DNA. In Dr. Jayaraman’s laboratory, two types of simulations were run: I. Atomistic molecular dynamics simulations and free energy calculations of multivalent ligands (based on spermines and lysines) binding to 12-15bp ds-DNA, and II. Development of coarse-grained models and simulations to study lysine based polymers binding to 100-1000bp ds-DNA. The key findings from the computational work done by PI Jayaraman and her group: a) Strong electrostatic interactions between the positively charged amines in the grafts and the negatively charged phosphates along the DNA backbone stabilizes bounds state. b) Core-grafted polyamine ligands bind to DNA with higher affinity than the free polyamines, in agreement with our hypothesis, due to larger enthalpic penalty for unbinding and lower entropic loss upon binding. This effect is most pronounced for the neomycin core ligands, which have six grafted spermines/ dilysines each. c) Spermine based ligands bind more strongly to DNA than dilysine based ligands due to differences in flexibility of the spermine and dilysine ligands. Major outcomes during the course of this project: PI Jayaraman and her graduate and undergraduate students have published one peer reviewed paper in Macromolecules http://pubs.acs.org/doi/full/10.1021/ma3011944 and have another paper in preparation for submission and review. This work was presented in the form of invited and contributed talks and poster at the AICHE Meeting in 2011, 2012 and 2013 by graduate student Robert Elder and PI Arthi Jayaraman. Broader Impacts: On the educational front, despite only ½ graduate student/year of financial support from this grant to PI Jayaraman, using some of her UColorado start up funds to supplement this grant, the PI trained 3 graduate students - R. Elder (PhD 2014), A. Van Fosson (MS 2014) and D. Johnson, and one undergraduate - Xiao Ba, (BS senior thesis ) on these computational tools. Robert Elder has found her a postdoctoral scientist position at the Army Research Laboratory since graduation. Outreach: PI Jayaraman was involved in outreach activities through the "Diversity action committee" at College of engineering, where summer bridge program and Gold Shirt programs are organized yearly to attract minority students into Engineering. PI Jayaraman also served as career panelist on the Gordon Research Seminar on Macromolecular Materials (2013) where she provided advice to graduate students and postdocs on a) various career paths in science and engineering, b) challenges faced by women in science and engineering and ways to overcome those challenges, and c) how to have a successful teaching and research career in academia.
