| 1. |
- Dias, Rita, et al.
(författare)
-
Electrophoretic properties of complexes between DNA and the cationic surfactant cetyltrimethylammonium bromide
- 2005
-
Ingår i: Electrophoresis. - : Wiley. - 0173-0835 .- 1522-2683. ; 26:15, s. 2908-2917
-
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.
|
|
| 2. |
- Bosaeus, Niklas, 1982, et al.
(författare)
-
Force-induced melting of DNA-evidence for peeling and internal melting from force spectra on short synthetic duplex sequences
- 2014
-
Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 42:12, s. 8083-8091
-
Tidskriftsartikel (refereegranskat)abstract
- Overstretching of DNA occurs at about 60-70 pN when a torsionally unconstrained double-stranded DNA molecule is stretched by its ends. During the transition, the contour length increases by up to 70% without complete strand dissociation. Three mechanisms are thought to be involved: force-induced melting into single-stranded DNA where either one or both strands carry the tension, or a B-to-S transition into a longer, still base-paired conformation. We stretch sequence-designed oligonucleotides in an effort to isolate the three processes, focusing on force-induced melting. By introducing site-specific inter-strand cross-links in one or both ends of a 64 bp AT-rich duplex we could repeatedly follow the two melting processes at 5 mM and 1 M monovalent salt. We find that when one end is sealed the AT-rich sequence undergoes peeling exhibiting hysteresis at low and high salt. When both ends are sealed the AT sequence instead undergoes internal melting. Thirdly, the peeling melting is studied in a composite oligonucleotide where the same AT-rich sequence is concatenated to a GC-rich sequence known to undergo a B-to-S transition rather than melting. The construct then first melts in the AT-rich part followed at higher forces by a B-to-S transition in the GC-part, indicating that DNA overstretching modes are additive.
|
|
| 3. |
- Bosaeus, Niklas, 1982, et al.
(författare)
-
Tension induces a base-paired overstretched DNA conformation
- 2012
-
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. ; 109:38, s. 15179-15184
-
Tidskriftsartikel (refereegranskat)abstract
- Mixed-sequence DNA molecules undergo mechanical overstretching by approximately 70% at 60-70 pN. Since its initial discovery 15 y ago, a debate has arisen as to whether the molecule adopts a new form [Cluzel P, et al. (1996) Science 271: 792-794; Smith SB, Cui Y, Bustamante C (1996) Science 271: 795-799], or simply denatures under tension [van Mameren J, et al. (2009) Proc Natl Acad Sci USA 106: 18231-18236]. Here, we resolve this controversy by using optical tweezers to extend small 60-64 bp single DNA duplex molecules whose base content can be designed at will. We show that when AT content is high (70%), a force-induced denaturation of the DNA helix ensues at 62 pN that is accompanied by an extension of the molecule of approximately 70%. By contrast, GC-rich sequences (60% GC) are found to undergo a reversible overstretching transition into a distinct form that is characterized by a 51% extension and that remains base-paired. For the first time, results proving the existence of a stretched basepaired form of DNA can be presented. The extension observed in the reversible transition coincides with that produced on DNA by binding of bacterial RecA and human Rad51, pointing to its possible relevance in homologous recombination.
|
|
| 4. |
|
|
| 5. |
- Carlsson, Nils, 1978, et al.
(författare)
-
Bicontinuous cubic phase of monoolein and water as medium for electrophoresis of both membrane-bound probes and DNA
- 2006
-
Ingår i: Langmuir. - : American Chemical Society (ACS). - 1520-5827 .- 0743-7463. ; 22:9, s. 4408-4414
-
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.
|
|
| 6. |
- Carlsson, Nils, 1978, et al.
(författare)
-
Diamond cubic phase of monoolein and water as an amphiphilic matrix for electrophoresis of oligonucleotides
- 2005
-
Ingår i: JOURNAL OF PHYSICAL CHEMISTRY B. ; 109:39, s. 18628-18636
-
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.
|
|
| 7. |
- Carlsson, Nils, 1978, et al.
(författare)
-
DNA hosted and aligned in aqueous interstitia of a lamellar liquid crystal - a membrane-biomacromolecule interaction model system
- 2013
-
Ingår i: Soft Matter. - : Royal Society of Chemistry (RSC). - 1744-6848 .- 1744-683X. ; 9:33, s. 7951-7959
-
Tidskriftsartikel (refereegranskat)abstract
- We report that DNA molecules can be intercalated and macroscopically oriented in the aqueous interstitia of a lyotropic lamellar liquid crystal. Using UV-vis linear dichroism and fluorescence spectroscopy we show that double-stranded oligonucleotides (25 base pairs) in the water-octanoate-decanol system remain base-paired in the B conformation and are confined in two dimensions, with the helix axis preferentially parallel to the lipid bilayer surfaces but free to rotate within this plane. The degree of helix confinement and the corresponding 2-D orientation can be improved by decreasing the thickness of the water interstitia via the fraction of water in the ternary mixture. Not surprisingly, the corresponding single-stranded oligonucleotides are not aligned, with their persistence length being short in comparison to the lamellar interstitium thickness. We propose this as a model system for studying interactions of DNA-ligand complexes near a lipid bilayer membrane which we demonstrate by using dye probes that are either covalently attached to one end of the oligonucleotide or reversibly bound by intercalation between the base pairs. Three cationic dyes, all strongly bound by intercalation to DNA when free in solution, are found to not bind to DNA but to prefer the membrane surface. The covalently attached Cy5 also binds to the bilayer while Cy3 tends to end-stack to the oligonucleotide duplex. The orientation of Cy5 parallel to the membrane indicates that electrostatic surface binding predominates over insertion into the hydrophobic interior of the membrane. Anionic and zwitterionic dyes (FAM and ROX) are found to remain randomly oriented in the water between the lipid bilayer surfaces. This journal is © The Royal Society of Chemistry.
|
|
| 8. |
- Carlsson, Nils, 1978, et al.
(författare)
-
Enzymes Immobilized in Mesoporous Silica: a Physical-Chemical Perspective
- 2014
-
Ingår i: Advances in Colloid and Interface Science. - : Elsevier BV. - 0001-8686. ; 205, s. 339-360
-
Forskningsöversikt (refereegranskat)abstract
- Mesoporous materials as support for immobilized enzymes have been explored extensively during the last two decades, primarily not only for biocatalysis applications, but also for biosensing, biofuels and enzyme-controlled drug delivery. The activity of the immobilized enzymes inside the pores is often different compared to that of the free enzymes, and an important challenge is to understand how the immobilization affects the enzymes in order to design immobilization conditions that lead to optimal enzyme activity. This review summarizes methods that can be used to understand how material properties can be linked to changes in enzyme activity. Real-time monitoring of the immobilization process and techniques that demonstrate that the enzymes are located inside the pores is discussed by contrasting them to the common practice of indirectly measuring the depletion of the protein concentration or enzyme activity in the surrounding bulk phase. We propose that pore filling (pore volume fraction occupied by proteins) is the best standard for comparing the amount of immobilized enzymes at themolecular level, and present equations to calculate pore filling from the more commonly reported immobilized mass. Methods to detect changes in enzyme structure upon immobilization and to study the microenvironment inside the pores are discussed in detail. Combining the knowledge generated from these methodologies should aid in rationally designing biocatalyst based on enzymes immobilized in mesoporous materials.
|
|
| 9. |
- Carlsson, Nils, 1978, et al.
(författare)
-
Quantification of protein concentration by the Bradford method in the presence of pharmaceutical polymers
- 2011
-
Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 411:1, s. 116-121
-
Tidskriftsartikel (refereegranskat)abstract
- We investigated how the Bradford assay for measurements of protein released from a drug formulation may be affected by a concomitant release of a pharmaceutical polymer used to formulate the protein delivery device. The main result is that polymer-caused perturbations of the Coomassie dye absorbance at the Bradford monitoring wavelength (595 nm) can be identified and corrected by recording absorptionspectra in the region of 350–850 mm. The pharmaceutical polymers Carbopol and chitosan illustrate two potential types of perturbations in the Bradford assay, whereas the third polymer, hydroxypropylmethylcellulose (HPMC), acts as a nonperturbing control. Carbopol increases the apparent absorbance at 595 nm because the polymer aggregates at the low pH of the Bradford protocol, causing a turbidity contribution that can be corrected quantitatively at 595 nm by measuring the sample absorbance at 850 nm outside the dye absorption band. Chitosan is a cationic polymer under Bradford conditions and interacts directly with the anionic Coomassie dye and perturbs its absorption spectrum, including 595 nm. In this case, the Bradford method remains useful if the polymer concentration is known but should be used with caution in release studies where the polymer concentration may vary and needs to be measured independently.
|
|
| 10. |
- Carlsson, Nils, 1978, et al.
(författare)
-
Spectroscopic characterization of Coomassie blue and its binding to amyloid fibrils
- 2012
-
Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 420:1, s. 33-40
-
Tidskriftsartikel (refereegranskat)abstract
- Coomassie brilliant blue G-250 (CB) is the dye used frequently in the Bradford assay for proteinconcentration determination. In this study, we investigated how the solvent polarity and viscosity affectthe CB absorption and fluorescence spectra and apply this understanding to investigate the binding of CBto lysozyme and insulin in the native and amyloid fibril states. Coomassie blue binds both to the nativeprotein and to amyloid fibrils but gives distinctly different spectral responses. The absorption andfluorescence spectra of CB indicate that binding sites in the fibrils are less polar and hold the CB dye morerigidly than in the native forms. The spectral comparison of CB bound to the two different fibrils showedthat the binding sites are different, and this was most likely due to differences in secondary structure asmonitored by circular dichroism. Finally, linear dichroism was used to show that the fibril-bound CB isoriented preferentially parallel to the insulin amyloid fibril axis.
|
|
