Capillary electrophoresis of nucleic acids has recently employed gels of self-assembled uncharged triblock copolymers as sieving media. Pluronic F127 contains poly(ethylene oxide) (EO) and poly(propylene oxide) (PO) with the block structure (EO)106(PO)70(EO)106. Aqueous solutions of 30% w/w of this polymer are liquids at low temperatures, but above 11 °C the polymers assemble to micelles that pack into a locally cubic lattice forming a gel-like lyotropic liquid crystal phase. Here we use linear dichroism spectroscopy to study the orientation dynamics of double-stranded DNA molecules during the electrophoresis. In 30% Pluronic F127, a 5400 bp DNA migrates with substantial perpendicular orientation of the helix axis, which is in contrast to electrophoresis in agarose gels where the helix axis of DNA is aligned parallel to the field direction. Comparison between linear and circular DNA indicates that neither DNA form enters the cubic microcrystals at low fields, and when combined with velocity measurements the kinetics of alignment buildup and relaxation suggests that migration instead occurs in grain boundaries between domains of microcrystals.

Electrophoretic properties of complexes between DNA and the cationic surfactant cetyltrimethylammonium bromideWe use agarose gel electrophoresis to characterize how the monovalent catioinic surfactant cetyltrimethylammonium bromide (CTAB) compacts double-stranded DNA, which is detected as a reduction in electrophoretic DNA velocity. The velocity reaches a plateau at a ratio R = 1.8 of CTAB to DNA-phosphate charges, i.e., above the neutralization point, and the complexes retain a net negative charge at least up to R = 200. Condensation experiments on a mixture of two DNA sizes show that the complexes formed contain only one condensed DNA molecule each. These CTAB-DNA globules were further characterized by time-resolved measurements of their velocity inside the gel, which showed that CTAB does not dissociate during the migration but possibly upon entry into the gel. Using the Ogston-model for electrophoresis of spherical particles, the measured in-gel velocity of the globule is quantitatively consistent with CTAB having two opposite effects, reduction of both the electrophoretic charge and DNA coil size. In the case of CTAB the two effects nearly cancel, which can explain why opposite velocity shifts (globule faster than uncomplexed DNA) have been observed with some catioinic condensation agents. Dissociation of the complexes by addition of anionic surfactants was also studied. The DNA release from the globule was complete at a mixing ratio between anionic and cationic surfactants equal to 1, in agreement with equilibrium studies. Circular DNA retained its supercoiling, and this demonstrates a lack of DNA nicking in the compaction-release cycle which is important in DNA transfection and purification applications. IntroductionComplexation with cationic lipids is one strategy for delivery of DNA to cells. Binding of the lipids leads to compaction of the DNA coils and to a reduction of its surface charge. Both effects are believed to contribute to the facilitated uptake of the nucleic acids through the cellular membrane [1][2][3][4][5]. Methods to monitor size and charge of the complexes are therefore helpful tools in the process of screening protocols for packaging the DNA before in vivo experiments are performed. Commonly a titration series is made where the ratio R between concentration of charges from cationic lipid and DNA-phosphate groups is varied, usually in a range that includes the point R = 1 where the number of added positive and negative charges is equal. It is widely accepted that surfactants display a very strong associative binding with DNA inducing its compaction [6][7][8], aggregation [9], and precipitation [8,10,11]. The associative phase behavior presented by these systems and the absence of redissolution with the addition of an excess of surfactant lead to the assumption that the formed complexes in solution are neutral [8,12].Electrophoresis has been used to study the properties of lipoplexes because the mobility reflects their net charge. Mobilities in free solution have been used [13,14] to q...

We investigate the dynamics of a protein-DNA complex under gel-shift assay conditions, in particular how the degree of gel confinement influences the complex stability during the electrophoretic analysis. The fiberlike complex between RecA proteins and a single-stranded oligonucleotide has been studied in hydroxyethylated agarose gels with the average pore radius being between 1 and 5 times larger than the radius of the rodshaped complex. The confining effect of the gel matrix slows down the dissociation of the complex, but in addition, migrative interactions (collisions) with the gel network during the electrophoresis perturbs the complex in two ways. At low gel concentrations, the protein-DNA complex is dismantled into free RecA and oligonucleotide with a half-time of the complex that decreases with increasing field strength in accordance with a migrative mechanism. At a given field strength, the half-time increases with increasing gel concentration despite more frequent interactions, probably because of a counteracting stabilizing cage effect from the gel. At high gel concentrations, a second type of perturbation is reflected in an increasing complex velocity over time, probably caused by trains of end-to-end attached fibers being broken up by interactions with the tight gels.

Lyotropic liquid crystals form highly regular porous matrices with aqueous channels on the nanometer length scale. We have used the cubic phases formed with water by either an amphiphilc block-copolymer (Pluronic F127) or by a lipid (monoolein) for electrophoretic separation of DNA and other biomolecules. Our goal is to use the well-defmed pores and the amphiphilic environment to obtain new separation motifs compared to conventional matrices, and to exploit the well-known phase diagrams ofthese two systems to optimise applications. The Pluronic crystal consists of close-packed micelles and its main advantage is that the cubic phase melts below 10°C, and we show that the separated DNA can be recovered in a biologically active state in preparative applications. Our mechanistic studies revealed that doublestranded DNA undergoes a highly non-conventional (non-reptative) mode of migration, with the helix axis perpendicular to the field direction because the DNA migrates in the grain boundaries of the polycrystalline samples. In contrast to the Pluronic case, the monoolein cubic crystal is bicontinuous, and a main advantage is that it is in equilibrium with a water-rich phase. We exploited this phase-behaviour in the useful sub-marine mode of analytical electrophoresis. The migration of oligonucleotides in the monoolein is strongly retarded compared to free solution and conventional gels, to an extent which is consistent with that migration indeed occurs through the nm-pores. We demonstrate separation of oligonucleotides based on size, and on different types of secondary structure of the same oligonucleotide size, such as the double-stranded, single-stranded and hairpin forms.