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The electrostatic binding of polycations with DNA‚EB complex results in displacement of intercalated cationic dye ethidium bromide (EB) from DNA double helix to the solution which is accompanied by a quenching of EB fluorescence. On the basis of this phenomenon, the fluorescence assay of DNA-containing polyelectrolyte complexes was recently developed. Data obtained in the current work demonstrate the applicability and advantages of this approach for monitoring both an interaction of DNA with cationic surfactants (CS) and stability of DNA-CS complexes. The comprehensive study was carried out with cationic detergents having different C12-C16 "tails" and "heads" with pyridinium or amino groups. In parallel, the similar experiments were performed with pyrenyl-tagged poly(methacrylate) anion (PMA*), in which the complex formation was monitored by quenching of PMA* fluorescence with pyridinium or nonquaternary amino groups of the detergents. The fluorescence titration curves of DNA‚EB or PMA* with CS consisted of two parts, with negligible quenching on the initial stage followed by the pronounced quenching. The critical aggregation concentration (CAC) determined from the intersection points of the curves decreased substantially with the length of "tail". CAC values measured in DNA-CS mixtures proved to be noticeably higher than those from PMA*-CS mixtures. This finding suggests that DNAinduced self-assembly of CS molecules in the intramacromolecular aggregates is hindered due to rigidity of the double helix. Dissociation of DNA-CS complexes in salt (NaCl) solutions was monitored by the increase of fluorescence intensity of EB intercalated in free sites of DNA. Inasmuch as the addition of salt resulted in increase of CAC and decrease of critical micelle concentration (CMC), two regimes of destruction of DNA-CS complexes dependent on CS concentration were revealed. The regime of noncooperative destruction was realized if CS concentration was lower than CMC at the ionic strength of the complex dissociation, otherwise the second regime of the cooperative destruction took place. In the latter case, the salt concentration corresponding to the destruction virtually did not depend on the length of "tail" but markedly decreased with increase of a number of N-methyl groups in the "head" in the series C12NH2 > C12NHMe > C12NMe2 > C12N + Me3Br -. It implies that distance between charges in the ion pairs is the dominant factor determining the stability of the complexes. In the case of a DNA-C12NMe2 complex, the destruction was rather pH sensitive and occurred at pH and ionic strength that were close to physiological conditions. The results might create the basis for design of DNA-CS complexes with controlled stability which could be of particular promise for DNA delivery to the target cell.

SUMMARY: Destruction of interpolyelectrolyte complexes (PEC) formed by DNA and different poly cations was achieved by addition of low molecular weight electrolytes. Monitoring of PEC dissociation was carried out by fluorescence quenching using the ability of the cationic dye ethidium bromide to intercalate into free sites of the DNA double helix accompanied by fluorescence. Degree of polymerization and charge density of the polycations as well as their N-alkyl substituentes (alkyl = methyl, ethyl, and propyl) were shown to be factors influencing the stability of PEC. The ability of added cations and anions to dissociate PEC decreases in the order Cawhich coincides with a decrease of affinity of the same counterions to DNA and to the polycation. The data obtained indicate that the change of the stability of DNA-containing PEC shows the same regularities as revealed for the stability of interpolyelectrolyte complexes formed by oppositely charged flexible synthetic polyelectrolytes in water-salt solutions.

Series of polycarboxybetaines (PCB-n) of pyridiniocarboxylate structure with the same degree of polymerization but differing in the number, n, of methylene groups in the alkyl spacer between charges in the betaine moieties, n = 1, 2, 3, 4, 5, and 8, were synthesized. The utility of PCB-n as positively charged components of polyelectrolyte complexes was elucidated by potentiometry, turbidimetry, and fluorescence spectroscopy. Affinity of PCB-n to the pyrenyl-tagged poly(methacrylic) acid (PMAA) or DNA was judged from the stability of the corresponding polyelectrolyte complexes in water-salt solutions at different pH values as monitored by fluorescence quenching techniques. At pH = 9.0, PCB-1 formed the least stable complexes due to strong interaction of charged groups positioned in close proximity in the betaine moieties. The increase in n resulted in the irregular change of the affinity. Thus, as expected, PCB-2 formed noticeably more stable complexes than PCB-1. However, PCB-3 and, in particular, PCB-4 revealed weaker affinity to PMAA or DNA that is attributed to formation of stable ion pairs between charges in the betaine rings. At neutral and slightly acidic pH values binding of all PCB-n except PCB-1 was drastically enhanced due to protonation of PCB-n carboxylic groups that occurred with a DeltapH shift of 2-3 units to higher values as compared with the protonation of free PCB-n. The ability of added polyanion to compete with the betaine carboxylic groups in binding with the pyridinium groups was supported by potentiometric titration of PCB-n mixtures with sodium poly(styrenesulfonate): for n > or = 2, the binding of the polyanion-competitor also shifted protonation of carboxylic groups to higher values with DeltapH of more than 2 units. Practical ramifications of the revealed role of the alkyl spacer in polyelectrolyte complexation as well as the pH-induced stabilization of the complexes that occurs under enzyme-friendly conditions might extend to areas of biotechnology, specifically in bioseparation and gene delivery.

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