Real-time Analysis of Plasmid DNA Binding Interactions with Immobilized Peptide Ligands using a Surface Plasmon Resonance Biosensor
Plasmid DNA for therapeutic and vaccination purposes must be highly purified. The high selectivity of affinity chromatography makes it ideal for the isolation of pDNA from complex biological feed stocks. Affinity makes use of the biological function and/or individual chemical structure of the interacting molecules. However, the success of any affinity purification protocol is dependent on the availability of suitable ligands. Surface plasmon resonance (SPR) has been employed for the detection and quantification of binding of the lac operon (lacO) sequence contained in a pDNA to synthetic peptides based on the DNA-binding domain of the lac repressor protein, lacI. These synthetic peptides are “biomimics” of the native lacI protein. In this research project, an assay was developed to allow the determination of the equilibrium dissociation constant (KD) and association and dissociation rate constants for the interaction between plasmid DNA and various peptide sequences, including a full lac binding region 47-mer, and a 27mer peptide, 16mer and 14mer peptide that represent sub-units of the full lac binding region. The KD values measured for the binding of pDNA to 47mer and 27mer peptide were 8.78±1.30 ×10-10 M and 7.21±0.56 ×10-10 M respectively while the KD values for 16-mer and 14mer peptide were 4.49±0.5 ×10-8 M and 6.22±0.89 ×10-6 M accordingly. These findings are highly relevant for the possible future use of these peptides as affinity ligands for the commercial purification of pDNA.