| 1. |
- Dias, Rita, et al.
(författare)
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Electrophoretic properties of complexes between DNA and the cationic surfactant cetyltrimethylammonium bromide
- 2005
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Ingår i: Electrophoresis. - : Wiley. - 0173-0835 .- 1522-2683. ; 26:15, s. 2908-2917
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Tidskriftsartikel (refereegranskat)abstract
- We 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 coll 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.
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| 2. |
- Carlsson, Nils, 1978, et al.
(författare)
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Bicontinuous cubic phase of monoolein and water as medium for electrophoresis of both membrane-bound probes and DNA
- 2006
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Ingår i: Langmuir. - : American Chemical Society (ACS). - 1520-5827 .- 0743-7463. ; 22:9, s. 4408-4414
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Tidskriftsartikel (refereegranskat)abstract
- Porous hydrogels such as agarose are commonly used to analyze DNA and water-soluble proteins by electrophoresis. However, the hydrophilic environment of these gels is not suitable for separation of important amphiphilic molecules such as native membrane proteins. We show that an amphiphilic liquid crystal of the lipid monoolein and water can be used as a medium for electrophoresis of amphiphilic molecules. In fact, both membrane-bound fluorescent probes and water-soluble oligonucleotides can migrate through the same bicontinuous cubic crystal because both the lipid membrane and the aqueous phase are continuous. Both types of analytes exhibit a field-independent electrophoretic mobility, which suggests that the lipid crystal structure is not perturbed by their migration. Diffusion studies with four membrane probes indicate that membrane-bound analytes experience a friction in the cubic phase that increases with increasing size of the hydrophilic headgroup, while the size of the membrane-anchoring part has comparatively small effect on the retardation.
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| 3. |
- Carlsson, Nils, 1978, et al.
(författare)
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Diamond cubic phase of monoolein and water as an amphiphilic matrix for electrophoresis of oligonucleotides
- 2005
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Ingår i: JOURNAL OF PHYSICAL CHEMISTRY B. ; 109:39, s. 18628-18636
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Tidskriftsartikel (refereegranskat)abstract
- We used a cubic liquid crystal formed by the nonionic monoglyceride monoolein and water as a porous matrix for the electrophoresis of oligonucleotides. The diamond cubic phase is thermodynamically stable when in contact with a water-rich phase, which we exploit to run the electrophoresis in the useful submarine mode. Oligonucleotides are separated according to size and secondary structure by migration through the space-filling aqueous nanometer pores of the regular liquid crystal, but the comparatively slow migration means the cubic phase will not be a replacement for the conventional DNA gels. However, our demonstration that the cubic phase can be used in submarine electrophoresis opens up the possibility for a new matrix for electrophoresis of amphiphilic molecules. From this perspective, the results on the oligonucleotides show that water-soluble particles of nanometer size, typical for the hydrophilic parts of membrane-bound proteins, may be a useful separation motif. A charged contamination in the commercial sample of monoolein, most likely oleic acid that arises from its hydrolysis, restricts useful buffer conditions to a pH below 5.6.
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| 4. |
- Cole, K. D., et al.
(författare)
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Single-molecule measurements of trapped and migrating circular DNA during electrophoresis in agarose gels
- 2006
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Ingår i: Electrophoresis. - : Wiley. - 0173-0835 .- 1522-2683. ; 27:22, s. 4396-4407
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Tidskriftsartikel (refereegranskat)abstract
- The effect of agarose gel concentration and field strength on the electrophoretic trapping of open (relaxed) circular DNA was investigated using microscopic measurements of individual molecules stained with a fluorescent dye. Three open circles with sizes of 52.5, 115, and 220 kbp were trapped by the electric field (6 V/cm) and found to be predominately fixed and stretched at a single point in the gel. The length of the stretched circles did not significantly change with agarose concentration of the gels (mass fractions of 0.0025, 0.01, and 0.02). The relaxation kinetics of the trapped circles was also measured in the gels. The relaxation of the large open circles was found to be a slow process, taking several seconds. The velocity and average length of the 52.5 kbp open circles and 48.5 kbp linear DNA were measured during electrophoresis in the agarose gels. The velocity increased when the agarose concentrations were lowered, but the average length of the open-circle DNA (during electrophoresis) did not significantly change with agarose gel concentrations. The circles move through the gels by cycles of stretching and relaxation during electrophoresis. Linear dichroism was also used to investigate the trapping and alignment of the 52.5 kbp open circles. The results in this study provide information that can be used to improve electrophoretic separations of circular DNA, an important form of genetic material and commonly used to clone DNA.
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| 5. |
- Eriksson, Maja, et al.
(författare)
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Binding of Intercalating and Groove-Binding Cyanine Dyes to Bacteriophage T5
- 2007
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Ingår i: The Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-6106 .- 1520-5207. ; 111:5, s. 1139-1148
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Tidskriftsartikel (refereegranskat)abstract
- The interaction between four related cyanine dyes and bacteriophage T5 is investigated with fluorescence and absorption spectroscopy. The dyes, which differ in size, charge, and mode of DNA-binding, penetrate the capsid and bind the DNA inside. The rate of association decreases progressively with increasing dye size, from a few minutes for YO to more than 50 h for YOYO (at 37 °C). The relative affinity for the phage DNA is a factor of about 0.2 lower than for the same T5-DNA when free in solution. Comparison of groove-bound BOXTO-PRO and intercalating YO-PRO shows that the reduced affinity is not due to DNA extension but perhaps influenced by competition with other cationic DNA-binding agents inside the capsid. Although, the extent of dye binding to the phages decreases with increasing external ionic strength, the affinity relative to free DNA increases, which indicates a comparatively weak screening of electrostatic interactions inside the phage. The rate of binding increases with increasing ionic strength, reflecting an increase in effective pore size of the capsid as electrostatic interactions are screened and/or a faster diffusion of the dye through the DNA matrix inside the capsid as the DNA affinity is reduced. A combination of electron microscopy, light scattering, and linear dichroism show that the phages are intact after YO-PRO binding, whereas a small degree of capsid rupture cannot be excluded with BOXTO-PRO.
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| 6. |
- Eriksson, Maja, 1975, et al.
(författare)
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Comparing mono- and divalent DNA groove binding cyanine dyes--Binding geometries, dissociation rates, and fluorescence properties
- 2006
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Ingår i: Biophysical Chemistry. - : Elsevier BV. - 1873-4200 .- 0301-4622. ; 122:3, s. 195-205
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Tidskriftsartikel (refereegranskat)abstract
- The unsymmetrical cyanine dyes BOXTO-PRO and BOXTO-MEE were derived from the DNA groove binder BOXTO, by adding a positively charged or a non-ionic hydrophilic tail to BOXTO, respectively. The main objective was to obtain more efficient DNA probes, for instance in electrophoresis and microscopy, by slowing down the dissociation of BOXTO from DNA. The interactions with mixed sequence DNA was studied with fluorescence and absorbance spectroscopy, stopped-flow dissociation and gel electrophoresis. Both the derivatives are groove bound as BOXTO, and have similar fluorescence properties when bound to mixed sequence DNA in free solution. BOXTO-PRO exhibits a slower dissociation than BOXTO from DNA, whereas the dissociation rate for BOXTO-MEE is faster and, unexpectedly independent of the ionic strength. During gel electrophoresis both BOXTO-PRO and BOXTO-MEE exhibit a faster dissociation rate than BOXTO. Still, BOXTO-PRO seems to be a good alternative as DNA probe, especially for applications in free solution where the dissociation is slower than for the corresponding intercalator TOPRO-1.
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| 7. |
- Eriksson, M., et al.
(författare)
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Time-resolved electrophoretic analysis of mobility shifts for dissociating DNA ligands
- 2005
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Ingår i: Electrophoresis. - : Wiley. - 0173-0835 .- 1522-2683. ; 26:3, s. 524-532
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Tidskriftsartikel (refereegranskat)abstract
- Intercalative binding of ligands to DNA can be demonstrated by helix unwinding, monitored by gel electrophoresis of supercoiled DNA, as electrophoretic mobility is sensitive to the topological DNA state. However, we show that an apparent lack of unwinding in an electrophoretic assay could be due to dissociation of the (intercalated) ligand during the analysis, rather than evidence for a nonintercalative mode of binding to DNA. Repetitive scanning during the electrophoresis ensures that release of the ligand during electrophoresis does not affect the measured degree of unwinding, based on the electrophoretic velocity being determined as a function of time. We use this assay to establish intercalation as a mode of binding to DNA for the cyanine dyes YO, YO-PRO as well as two enantiomeric forms of the ruthenium complexes [(phen)2 Ru(tatpp)Ru(phen)2]4+, and to support groove-binding for the new unsymmetrical cyanine dyes BOXTO and BOXTO-PRO. Groove-binding could be concluded from a lack of unwinding, because we could rule out that it is caused by release of the dye during the electrophoresis. The gel electrophoresis has the advantage over hydrodynamic techniques that much smaller sample amounts are required, and our time-resolved approach can be employed in all mobility-shift assays when applied to dissociating complexes.
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| 8. |
- Sanandaji, Nima, 1981, et al.
(författare)
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Comparison of oligonucleotide mirgation in a bicontinuous cubic phase of monoolein and water and in a fibrous agarose hydrogel
- 2006
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Ingår i: Electrophoresis. - : Wiley. - 1522-2683 .- 0173-0835. ; 27:15, s. 3007-3017
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Tidskriftsartikel (refereegranskat)abstract
- Porous hydrogels such as agarose are commonly used to analyze DNA and water-soluble proteins by electrophoresis. More recently lyotropic liquid crystals, such as the diamond cubic phase formed by the lipid monoolein and water, has become a new type of well-defined porous structure of interest for both hydrophilic and amphiphilic analytes. Here we compare these two types of matrixes by investigating the nature of retardation they confer to an oligonucleotide that migrates in their respective aqueous phases. The retardation for a 25-mer oligonucleotide was found to be about 35-fold stronger in the cubic phase than in an agarose hydrogel modified to have the same average pore size. According to modelling, the strong retardation is primarily due to the fact that hydrodynamic interaction with the continuous monoolein membrane is a stronger source of friction than the steric interactions (collisions) with discrete gel fibres. A secondary effect is that the regular liquid crystal has a narrower pore-size distribution than the random network of the agarose gel. In agreement with experiments, these two effects together predict that the retardation in the cubic phase is a 30-fold stronger than in an agarose gel with the same average pore radius.
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| 9. |
- Tokarz, Michal, 1976, et al.
(författare)
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Electrophoretic transport of latex particles in lipid nanotubes
- 2007
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Ingår i: Langmuir. - : American Chemical Society (ACS). - 1520-5827 .- 0743-7463. ; 23:14, s. 7652-7658
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Tidskriftsartikel (refereegranskat)abstract
- Lipid vesicles can be connected by membrane nanotubes to build networks with promising bioanalytical properties. Here we characterize electrophoretic transport in such membrane tubes, with a particular eye to how their soft-material nature influences the intratube migration. In the absence of field, the tube radius is 110 +/- 26 nm, and it remains in this range during electrophoresis even though the applied electric field causes a slight decrease in the tube radius (similar to 6-11%). The electrophoretic velocity of the membrane wall (labeled with quantum dots) varies linearly with the field strength. Intratube migration is studied with latex spheres of radii 15, 50, 100, and 250 nm. The largest particle size does not enter the tube at fields strengths lower than 1250 V/m because the energy cost for expanding the tube around the particles is too high. The smaller particles migrate with essentially the same velocity as the membrane at low fields. Above 250 V/cm, the 15 nm particles exhibit an upward deviation from linear behavior and in fact migrate faster than in free solution whereas the 100 nm particles deviate downward. We propose that these nonlinear effects arise because of lipid adsorption to the particles (dominating for 15 nm particles) and a pistonlike compression of the solvent in front of the particles (dominating for 100 nm). As expected from such complexities, existing theories for a sphere migrating in a rigid-wall cylinder cannot explain our velocity results in lipid nanotubes.
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| 10. |
- Tokarz, Michal, 1976, et al.
(författare)
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Single-file electrophoretic transport and counting of individual DNA molecules in surfactant nanotubes
- 2005
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 102:26, s. 9127-9132
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Tidskriftsartikel (refereegranskat)abstract
- We demonstrate a complete nanotube electrophoresis system (nanotube radii in the range of 50 to 150 nm) based on lipid membranes, comprising DNA injection, single-molecule transport, and single-molecule detection. Using gel-capped electrodes, electrophoretic single-file transport of fluorescently labeled dsDNA molecules is observed inside nanotubes. The strong confinement to a channel of molecular dimensions ensures a detection efficiency close to unity and identification of DNA size from its linear relation to the integrated peak intensity. In addition to constituting a nanotechnological device for identification and quantification of single macromolecules or biopolymers, this system provides a method to study their conformational dynamics, reaction kinetics, and transport in cell-like environments.
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