Binding of Self-assembling Peptides to Oligodeoxynucleotides
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This thesis is an experimental investigation on the binding of self-assembling peptides to oligodeoxynucleotides (ODNs) and the characterization of the resulting peptide-ODN complexes/aggregates, the first key step in the development of a peptide-based gene delivery system. Effects of pH, charge distribution along the peptide backbone, and oligonucleotide sequences on the peptide-ODN binding were investigated by a series of physicochemical methods. UV-Vis absorption and fluorescence anisotropy experiments demonstrate that aggregates are formed after mixing the peptide and ODN in aqueous solution. The aggregates in solution can be centrifuged out. Based on this property, the fraction of ODNs incorporated in the peptide-ODN aggregates can be obtained by comparing the UV-Vis absorption of the solution before and after centrifugation. Binding isotherms are generated by a binding density function analysis of the UV absorbance results. The binding parameters are extracted from the analysis of the binding isotherms based on the McGhee and von Hippel model. Equilibrium binding parameter studies show that the binding of two self-assembling peptides, EAK16-II and EAK 16-IV, to model single and double-stranded ODNs at pH 4 is stronger than at pH 7, and that no binding occurs at pH 11. These results demonstrate that electrostatic interactions play an important role in the EAK-ODN binding because EAKs are more positively charged at low pH. EAKs bind more strongly to dG16 than to the other ODN sequences dC16 and dGC16. This demonstrates that the hydrogen bond might be involved because they promote the binding of the lysine residues of the peptide to dG16 to a greater extent than to dC16. The charge distribution along the peptides is found to have an effect on the binding. EAK16-IV, whose positively charged residues are clustered at one end of the peptide, binds to the ODNs more strongly than EAK16-II, whose positively charged residues are distributed throughout the peptide chain, at the same pH. The binding process of EAKs to the ODNs was investigated by fluorescence anisotropy and static light scattering experiments. The results show that individual EAK and ODN molecules complex first, followed by the aggregation of these complexes into large aggregates. The nature of the resulting peptide-ODN complexes/aggregates is examined by UV-Vis absorption, fluorescence anisotropy, and PAGE experiments. The results demonstrate that free EAK, free ODNs, and small EAK-ODN complexes, which can not be centrifuged out, exist in the supernatant, and that large aggregates are collected in the pellets after centrifugation of the solution. The size of the resulting EAK-ODN complexes/aggregates measured by AFM and DLS is around a few hundreds of nanometers at low EAK concentrations. The accessibility of the ODNs to the quencher in the solution is reduced by 40 % and 60 % after binding to EAK16-II and EAK16-IV, respectively, as determined by fluorescence quenching experiments on EAK-ODN mixture solutions. An ODN protection from Exonuclease 1 degradation is provided by the EAK16-II or EAK16-IV matrix when they are mixed with the ODNs at pH 4. However, the ODNs are protected to a much lower degree when the EAK-ODN aggregates are prepared at pH 7. The EAK-ODN aggregates prepared at pH 7 are found to dissociate more easily than those prepared at pH 4 when they are incubated with exonuclease I solution at pH 9.5. These results suggest that the ODN protection afforded by the EAK-ODN aggregates is correlated with their structural stability after being incubated with the nuclease solution. The stability of the EAK-ODN aggregates after dilution is determined by UV-Vis absorption. No detectable dissociation of the aggregates is observed over 20 hrs after a 5- and 10-fold dilution of the solution in the same buffer used for their preparation. The EAK-ODN aggregates remain stable after the solutions are centrifuged, and re-dissolved in fresh buffer solutions. The ability of an EAK matix to protect ODNs from nuclease degradation together with its biocompatibility and low-toxicity suggests that EAK self-assembling peptides could be used as carriers for gene delivery.