Surfactant microstructures as DNA sequence tags

We have developed surfactant microstructures, such as liposomes and micelles, for signal amplification of specific nucleic acid sequences in biosensing applications. These surfactant structures bind target strands in solution, which has kinetic and thermodynamic advantages over nucleic acid hybridization at solid-liquid interfaces. Attractive hydrophobic interactions between target nucleobases and surfactant structures can counteract the electrostatic repulsion and steric effects experienced by target strands on solid surfaces, allowing for hybridization of longer sequences.; We covalently attached peptide nucleic acid (PNA) to liposomes by conjugation of PNA sequences to synthetic di-alkyl lipids and co-extrusion with helper lipids. PNA is an uncharged DNA analog with superior DNA binding properties. To enhance the aqueous solubility of PNA amphiphiles (PNAA), we experimented with a number of charged amino acid residues and found that the addition of four glutamic acid residues and two ethylene oxide spacers was required to properly hydrate 10-mer PNAA.; The extent of PNA/DNA hybridization on liposomes was assessed by capillary zone electrophoresis. We found that nearly all PNA sequences on the surface were hybridized to a stoichiometric amount of same-length (10-mer), complementary DNA. PNA/DNA hybridization was not observed when we introduced a single-base mismatch in the DNA sequence. DNA containing distal overhanging bases showed attenuated binding efficiency, likely due to electrostatic repulsion between the PNA liposome double-layer and the DNA backbone. PNA liposomes were able to bind DNA with proximal overhanging bases by compensating for electrostatic repulsion with nonspecific attraction between nucleobases and the lipid bilayer. We developed a model based on Gouy-Chapman theory to calculate the levels of ionic strength at which the free energy of PNA/DNA hybridization would be able to overcome electrostatic repulsion.; We synthesized fluorophore-labeled PNAA to examine the partitioning of PNA/DNA duplexes in lipid bilayers. We found that PNA/DNA duplexes with DNA sequences containing up to 20 bases remain in the lipid structures. When the DNA sequence length was 40 bases, PNA/DNA extraction was observed. When the sequence length was 60 bases, it appeared that electrostatic repulsion prevented hybridization.; We also studied PNA/DNA hybridization on micellar structures. Although mono-alkyl PNAA were extracted from micelles upon DNA hybridization, we were able to retain PNA/DNA duplexes with di-alkyl PNAA. By using a laser-induced fluorescence detector, we were able to reduce the detection limit of our system by three orders of magnitude, to 10--100 nM.