Sequence-specific DNA purification using peptide nucleic acid amphiphiles

We have developed a novel DNA purification process utilizing hydrophobic affinity tags specifically tailored to recognize pre-determined nucleic acid sequences for the efficient and rapid recovery of DNA samples from impure samples. Peptide nucleic acids (PNAs) were used as DNA capture strands with monoalkyl hydrocarbons appended to the N-terminus of the peptide creating a PNA-amphiphile (PNAA). PNA acts as a DNA mimic allowing for hybridization to complementary DNA and RNA targets with greater specificity and thermal stability due to the replacement of the phosphodiester backbone with neutral peptide linkages. These characteristics allow PNA to recognize both single stranded (ss) and double-stranded (ds) DNA targets. A unique strand invasion mechanism forming (PNA)2/DNA triplexes facilitates the development of purification methods aimed at recovering double stranded DNA samples including PCR products, cell extract targets and plasmids carrying unique gene sequences.; Since PNAs posses an uncharged backbone aqueous solubility is somewhat limited, appending a hydrophobic lipid tail to the N-terminus will only act to limit solubility even further. This presents a potential problem for solution based purification methods. As such, efforts were made to enhance the aqueous solubility of PNAAs by examining the effects of tail length/architecture, sequence length and incorporation of charged amino acid residues. PNAA solubility was found to be a strong function of buffer type and pH. Structural properties have been correlated to observed melting temperatures for PNAA/DNA duplexes. Amino acid charge was found to affect stability through electrostatic interactions with the DNA backbone while the lipid tail did not alter duplex melting temperatures. Overhanging DNA bases on the binding target were found to have a stabilizing effect on duplex stability.; Using hydrophobic interaction chromatography (HIC) we have demonstrated that PNAA/DNA duplexes can be separated from unbound DNA and other components with high resolution and sequence specificity without the use of stringency steps to remove any noncomplementary targets. By optimizing PNAA/DNA ratio and hybridization time, separation (capture) efficiencies of 96% were observed. PNAAs readily mix with cosurfactants such as sodium dodecylsulfate (SDS) to form PNAA mixed micelles. (Abstract shortened by UMI.)...