Sequence-Specific Binding of DNA to Liposomes Containing Di-Alkyl Peptide Nucleic Acid (PNA) Amphiphiles Bruno F. Marques and James W. Schneider* Department of Chemical Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213-3890 Received August 13, 2004. In Final Form: November 24, 2004 We present a method to covalently attach peptide nucleic acid (PNA) to liposomes by conjugation of PNA peptide to charged amino acids and synthetic di-alkyl lipids (“PNA amphiphile,” PNAA) followed by co- extrusion with disteroylphosphatidylcholine (DSPC) and cholesterol. Attachment of four Glu residues and two ethylene oxide spacers to the PNAA was required to confer proper hydration for extrusion and presentation for DNA hybridization. The extent of DNA oligomer binding to 10-mer PNAA liposomes was assessed using capillary zone electrophoresis. Nearly all PNAs on the liposome surface are complexed with a stoichiometric amount of complementary DNA 10-mers after 3-h incubation in pH 8.0 Tris buffer. No binding to PNAA liposomes was observed using DNA 10-mers with a single mismatch. Longer DNA showed a greatly attenuated binding efficiency, likely because of electrostatic repulsion between the PNAA liposome double layer and the DNA backbone. Langmuir isotherms of PNAA:DSPC:chol monolayers indicate miscibility of these components at the compositions used for liposome preparation. PNAA liposomes preserve the high sequence-selectivity of PNAs and emerge as a useful sequence tag for highly sensitive bioanalytical devices. Introduction While the use of liposomes as model membranes and in drug and gene delivery applications is well appreciated, their encapsulation properties can also be applied to the creation of highly sensitive bioanalytical devices. A 1000- fold signal amplification can be achieved using liposomes filled with fluorescent material as probes. 1,2 Signal amplification can also be carried out with nonfluorescent liposomes by dendritic amplification, where networks of specifically linked liposomes are bound to surfaces. These networks can be detected by Faradaic impedance spec- troscopy or by microgravimetry, with detection limits as low as 10 -13 M. 3,4 Other biosensing applications rely on the release of electrochemical indicators from the core of liposomes 5-7 or colorimetric transitions of polydiacetylene amphiphiles in the bilayer. 8 Most recently, liposomes have been implemented in microfluidic chips for biosensing purposes. 9, 10 The unique binding properties of peptide nucleic acids (PNA) give them important advantages in biosensing applications. These synthetic nucleic acid analogues bind complementary DNA to form a PNA-DNA duplex that is more stable than the corresponding DNA-DNA duplex. 11 PNA is a structural mimic of DNA that replaces the negatively charged sugar-phosphate backbone of DNA with an uncharged N-(2-aminoethyl)glycine backbone, and the added stability of PNA-DNA duplexes has been ascribed to the lower degree of charge repulsion for PNA- DNA duplex formation. 12,13 PNA-DNA duplex stabilities are highly sensitive to single-base mismatches. 14 PNA can also bind specific dsDNA targets by triplex formation, even in biological buffers that suppress triplex formation between ssDNA and dsDNA. 15,16 By constructing liposomes hosting PNA in an active form, we should be able to achieve the highly sensitive detection levels afforded by liposomes while adding the highly selective binding properties of PNA. A difficulty encountered when working with PNAs is that they are sparingly soluble in water and have a tendency to self-aggregate in solution. While these effects have not been studied extensively, PNA oligomers are soluble in water at concentrations below about 10 μM, and their solubility is improved by the addition of a terminal lysine group. 17 To better understand their cellular uptake, Wittung et al. measured low rates of efflux of PNA oligomers from the interior of liposomes, indicating that their solubility in the nonpolar lipid bilayer is also fairly low. 18 Hence, the low solubility of PNA in both polar * To whom correspondence should be addressed. (1) Lee, M.; Durst, R. A.; Wong, R. B. Anal. Chim. Acta 1997, 354, 23-28. (2) Yap, W. T.; Locascio-Brown, L.; Plant, A. L.; Choquette, S. J.; Horvath, V.; Durst, R. A. Anal. Chem. 1991, 63, 2007-11. (3) Patolsky, F.; Lichtenstein, A.; Willner, I. Angew. Chem., Int. Ed. 2000, 39, 940-943. 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