| 21. |
- Gisselfält, Katrin, 1969, et al.
(author)
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Interactions of tris(phenanthroline)ruthenium(II) enantiomers with DNA: Effects on helix flexibility studied by the electrophoretic behavior of reptating DNA in agarose gel
- 2000
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In: Journal of Physical Chemistry B Materials. - : American Chemical Society (ACS). - 1089-5647 .- 1520-6106 .- 1520-5207. ; 104:15, s. 3651-3659
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Journal article (peer-reviewed)abstract
- A combination of measurement of mobility and orientational dynamics of long reptating DNA in agarose gel has been used to reveal how the binding of Delta and Lambda enantiomers of the tris(phenanthroline)ruthenium(II) ion affects the flexibility of the DNA helix. The mobility data, and data over the step length and period time of the reptation cycle, obtained in the presence of the respective enantiomer, are compared with those of free DNA and with data obtained earlier on DNAs with known variations in helix flexibility. The results suggest that the Delta form induces kinks in the DNA helix while the Lambda form gives rise to a local stiffening of the helix.
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| 22. |
- Hagelin, Christina, 1977, et al.
(author)
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A polarized-light spectroscopy study of interactions of a hairpin polyamide with DNA
- 2006
-
In: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 91:3, s. 904-911
-
Journal article (peer-reviewed)abstract
- We here study the interactions of a polyamide with large DNA, and compare to those of minor groove binder distamycin (DST), including high ligand/DNA binding ratios. Specific as well as nonspecific binding is probed using polarized-light spectroscopy combined with singular value decomposition analysis. Circular and linear dichroism data confirm binding geometries consistent with minor groove binding for both of the ligands. Interestingly, at high and intermediate ligand/DNA ratios the polyamide exhibits no significant sequence discrimination between mixed-sequence (calf thymus) and AT DNA as compared to DST. Each ligand is concluded to exhibit two different binding modes depending upon ligand/DNA ratio and nucleo-base sequence. At high binding ratios, distinct differences between the ligands are observed: circular dichroism spectra exciton effects provide evidence of bimolecular interactions of the polyamide when bound to AT-DNA, whereas no effects are seen with DST or mixed-sequence DNA. Also linear dichroism indicates that a change in binding geometry occurs at high polyamide/AT ratios, and that the effect occurs only with polyamide in contrast to DST. Since the effect is insignificant with DST, or with calf thymus DNA, it is concluded that it relates to the sizes of the ligands and the minor grooves, becoming critical in the limit of crowding.
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| 23. |
- Hagelin, Christina, 1977, et al.
(author)
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Assigning Membrane Binding Geometry of Cytochrome c by Polarized Light Spectroscopy
- 2009
-
In: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 96:8, s. 3399-3411
-
Journal article (peer-reviewed)abstract
- In this work we demonstrate how polarized light absorption spectroscopy (linear dichroism (LD)) analysis of the peptide ultraviolet-visible spectrum of a membrane-associated protein (cytochrome (cyt) c) allows orientation and structure to be assessed with quite high accuracy in a native membrane environment that can be systematically varied with respect to lipid composition. Cyt c binds strongly to negatively charged lipid bilayers with a distinct orientation in which its a-helical segments are on average parallel to the membrane surface. Further information is provided by the LID of the pi-pi* transitions of the heme porphyrin and transitions of aromatic residues, mainly a single tryptophan. A good correlation with NMR data was found, and combining NMR structural data with LID angular data allowed the whole protein to be docked to the lipid membrane. When the redox state of cyt c was changed, distinct variations in the LID spectrum of the heme Soret band were seen corresponding to changes in electronic transition energies; however, no significant change in the overall protein orientation or structure was observed. Cyt c is known to interact in a specific manner with the doubly negatively charged lipid cardiolipin, and incorporation of this lipid into the membrane at physiologically relevant levels was indeed found to affect the protein orientation and its a-helical content. The detail in which cyt c binding is described in this study shows the potential of LID spectroscopy using shear-deformed lipid vesicles as a new methodology for exploring membrane protein structure and orientation.
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| 24. |
- Hagelin, Christina, 1977, et al.
(author)
-
Membrane interactions of cell-penetrating peptides probed by tryptophan fluorescence and dichroism techniques: Correlations of structure to cellular uptake
- 2006
-
In: Biochemistry. - : American Chemical Society (ACS). - 1520-4995 .- 0006-2960. ; 45:24, s. 7682-7692
-
Journal article (peer-reviewed)abstract
- This work reports on the binding and conformation of a series of CPPs in the bilayer membranes of large unilamellar vesicles and the effect of the presence of cholesterol. We show a negative correlation between alpha-helical structure and uptake efficiency for penetratin peptides where the two central arginine residues of penetratin are thought to be important for breaking the secondary structure. Penetratin alpha-helicity is also reduced upon incorporation of cholesterol into the membrane. Flow linear dichroism in the far-UV region shows that the penetratin peptides adopt a preferential orientation of the alpha-helix parallel to the bilayer, and the linear dichroism (LD) spectrum in the aromatic region indicates that the tryptophan residues are preferentially oriented parallel to the membrane. The Tat analogue TatP59W and the oligoarginine R7W, which are more efficient CPPs than penetratin, bind to membranes as random coils and do not show any orientation in LD, again indicating that alpha-helicity reduces uptake efficiency. Further, we observe large variations in tryptophan quantum yields for the five CPPs in this study and discuss this in terms of the ability to cause lipid rearrangement. Binding isotherms show that cholesterol increases the affinity of the peptide for the membrane, but tryptophan fluorescence lifetimes are essentially unaltered by incorporation of as much as 40 mol % cholesterol into the membrane, suggesting the absence of specific peptide-cholesterol interactions. Fluorescence emission maxima are insensitive to cholesterol and indicate that the peptide is positioned in the headgroup region. The results on peptide-membrane interactions are discussed in terms of possible uptake mechanisms.
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| 25. |
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| 26. |
- Höök, Fredrik, 1966, et al.
(author)
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Characterization of PNA and DNA Immobilization and Subsequent Hybridization with DNA Using Acoustic-Shear-Wave Attenuation Measurements
- 2001
-
In: Langmuir. - : American Chemical Society (ACS). - 1520-5827 .- 0743-7463. ; 17:26, s. 8305-8312
-
Journal article (peer-reviewed)abstract
- We report here how the quartz crystal microbalance with dissipation monitoring (QCM-D) technique, simultaneously measuring changes in the induced energy dissipation, D (cf. viscoelastic properties), and the frequency, f (cf. coupled mass), can be used to characterize the bound state of single-stranded peptide nucleic acid (PNA) and deoxyribose nucleic acid (DNA) in relation to their ability to function as selective probe(s) for fully complementary and single-mismatch DNA. The possibility to use the QCM-D technique for detection of binding kinetics and structural differences in the formed duplexes is also presented. We found that thiol-PNA and thiol-DNA attached via a sulfur group directly on a bare-gold surface are less efficient as probes for DNA than are biotin-PNA and biotin-DNA, coupled on top of a two-dimensional (2-D) arrangement of streptavidin, formed on a biotinylated phospholipid bilayer on a SiO2 surface. The fully complementary and singly mismatched DNA oligomers hybridize with the immobilized PNA and DNA. A single mismatch is discriminated via a significant difference in the binding and dissociation kinetics, demonstrating a high selectivity and thus successful immobilization of functional single strands. The observed ratios between hybridization-induced energy dissipation (DeltaD) and the frequency shift (Deltaf) made it possible to discriminate thiol-PNA directly attached to a gold surface from biotin-PNA coupled to the streptavidin 2-D arrangement, where the former were shown to be inefficient for detecting subsequent hybridization. Structural differences of the immobilized layers composed of biotin-PNA-DNA and biotin-DNA-DNA were clearly reflected by the DeltaD and Deltaf response.
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| 27. |
- James, PL, et al.
(author)
-
Effects of a hairpin polyamide on DNA melting: comparison with distamycin and Hoechst 33258
- 2004
-
In: Biophysical Chemistry. - : Elsevier BV. - 1873-4200 .- 0301-4622. ; 111:3, s. 205-212
-
Journal article (peer-reviewed)abstract
- We have used DNase I footprinting and fluorescence melting studies to study the interaction of the hairpin polyamide Im-Py-Py-Py-(R)(H2N)gamma-Im`-PY-PY-PY-beta-Dp with its preferred binding sites (5'-WGWWCW; W=A or T) and other sequences. DNase I footprinting confirmed that the ligand binds to the sequence AGAACA at nanomolar concentrations and that changing the terminal A to G causes a dramatic decrease in affinity, while there was no. interaction with the reverse sequence WCWWGW Fluorescence melting studies with I I mer duplexes showed that the polyamide had very different effects on the forward (TGWWCT) and reverse (TCTAGT) sequences. At low concentrations, the polyamide produced biphasic melting curves with TGATCT, TGTACT and TGAACT, suggesting a strong interaction. In contrast, the melting profiles with TCTAGT were always monophasic and showed much smaller concentration dependent changes in T-m. The polyamide also showed weak binding to the sequence TGATCT when one of the central AT pairs was replaced with an AC mismatch. These melting profiles were compared with those produced by the AT-selective minor groove binding agents distamycin and Hoechst 33258 at the same site's and at similar sequences containing A(5) and (AT)(3), which are expected to bind distamycin in the 1:1 and 2:1 modes, respectively. These ligands produced simple monophasic melting curves in which the T-m steadily increased as the ligand concentration was raised. (C) 2004 Elsevier B.V. All rights reserved.
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| 28. |
- Kim, Hye-Kyung, 1970, et al.
(author)
-
ADP Stabilizes the Human Rad51-single stranded DNA Complex and Promotes Its DNA Annealing Activity
- 2002
-
In: Genes to Cells. - : Wiley. - 1356-9597 .- 1365-2443. ; 7:11, s. 1125-1134
-
Journal article (peer-reviewed)abstract
- Background: Human Rad51 protein (HsRad51) is a homologue of Escherichia coli RecA protein, and involved in homologous recombination. These eukaryotic and bacterial proteins catalyse strand exchange between two homologous DNA molecules, each forming a complex with single-stranded DNA (ssDNA) and ATP as the initial step. Both proteins hydrolyse ATP; however, the role of ATP hydrolysis appears to vary between the two proteins.Results: Measurements using the fluorescence ssDNA analogue, poly(1,N (6) -etheno-deoxyadenosine), indicate that ATP affects the HsRad51-ssDNA complex, promoting two conformational states: one transient, rather rigid transition state and a final more flexible state. While ADP lowers the affinity of RecA protein to ssDNA, it is found to rather stabilize the HsRad51-ssDNA complex. ADP does not activate the strand exchange by HsRad51 but instead stimulates annealing between complementary ssDNAs.Conclusions: The hydrolysis of ATP promotes a transition of the HsRad51-ssDNA complex from a stiff state to less stiff state. The first state may be important for the strand separation of dsDNA in the initial step of strand exchange, while the second state may be important for annealing in the next step. However, hydrolysis does not dissociate HsRad51 from DNA as a component step of its recycling.
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| 29. |
- Kim, Hye-Kyung, 1970, et al.
(author)
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Co-Ion Dependence of DNA Nuclease Activity Suggests Hydrophobic Cavitation as a Potential Source of Activation Energy
- 2001
-
In: European Physical Journal E. - : Springer Science and Business Media LLC. - 1292-8941 .- 1292-895X. ; 4:4, s. 411-417
-
Journal article (peer-reviewed)abstract
- The source of the activation energy that allows cutting of DNA by restriction enzymes is unclear. A systematic study of the cutting efficiency of the type-II restriction endonuclease EcoRI, with varying background electrolyte ion pair and buffer reported here, shows only a modest dependence of efficiency on cation type. Surprisingly, efficiency does depend strongly on the presumed indifferent anion of the background salt. What emerges is that competition between the background salt anion and the buffer anion for the enzyme and DNA surfaces is crucial. The results are unexpected and counterintuitive from the point of view of conventional electrolyte theory. However, taken together with recent developments in surface chemistry, the results do fall into place and could also suggest a novel mechanism for enzyme activity as an alternative to metal-activated hydrolysis: microscopic cavitation in a hydrophobic pocket might be the source of activation energy.
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| 30. |
- Lundberg, Erik, 1981, et al.
(author)
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Addressable molecular node assembly - high information density DNA nanostructures
- 2008
-
In: Nucleic acids symposium series (2004). - 1746-8272. ; :52, s. 683-684
-
Journal article (peer-reviewed)abstract
- The inherent self-assembly properties of DNA make it ideal in nanotechnology. We present a fully addressable DNA nanostructure with the smallest possible unit cell, a hexagon with a side-length of only 3.4 nm.(2,3) Using novel three-way oligonucleotides, where each side has a unique double-stranded DNA sequence that can be assigned a specific address, we will build a non-repetitive two-dimensional grid.
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