Innovations in gene therapy have brought researchers closer to finding a cure for genetic disorders through the regulation of gene expression. For treatment via gene therapy, one must transfect nucleic acids into the cell, a process made more efficient by the use of  delivery agents. Cationic polymers, such as polyamidoamine (PAMAM) dendrimer, polyethyleneimine (PEI), and transactivating transcription factor (TAT) peptide of HIV-1, are attractive agents due to their ease of synthesis, nucleic acid loading capacity and lower immunogenicity compared to viral vectors, The binding of DNA to these delivery agents is vital to the efficiency of gene delivery. To study the effect of DNA structure on its binding to the three delivery agents above, ethidium bromide exclusion assays, electrophoretic mobility shift assays (EMSA), and isothermal titration calorimetry (ITC) were used to determine binding affinity and thermodynamics. From ethidium bromide exclusion assay, it has been found that single- and double-stranded DNA bind similarly and stronger than plasmid DNA. However, ITC data shows plasmid DNA binds much stronger (one order of magnitude) than both double- and single-stranded DNA for all three delivery agents. PAMAM and PEI, polycations of similar molecular weight but different architecture, bind similarly to all types of DNA and both stronger than TAT peptide. This affinity is likely a result of positive cooperativity of the larger polymer structures. Poly(A), poly(T), and poly(C) homonucleotide sequences show no interaction with any polymer, leading to interesting conclusions about the effect of sequence specificity on binding, which is being studied further. Circular dichroism (CD) spectroscopy will also be used to understand secondary structure changes that occur when different types of DNA bind various delivery agents. This data can be used to determine the most effective combination of delivery agent for different structures and sequences of DNA for transfecting cells during gene therapy.

Effect of DNA structure on its binding to polycationic delivery agents

247th National Meeting (2014)

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