| 51. |
- Persson, Daniel, 1972, et al.
(author)
-
Vesicle size-dependent translocation of penetratin analogs across lipid membranes
- 2004
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In: Biochimica et Biophysica Acta - Biomembranes. - : Elsevier BV. - 1879-2642 .- 0005-2736. ; 1665:1-2, s. 142-155
-
Journal article (peer-reviewed)abstract
- The recent discoveries of serious artifacts associated with the use of cell fixation in studies of the cellular uptake of cell-penetrating peptides (CPPs) have prompted a reevaluation of the current understanding of peptide-mediated cellular delivery. Following a report on the differential cellular uptake of a number of penetratin analogs in unfixed cells, we here investigate their membrane translocation abilities in large and giant unilamellar vesicles (LUVs and GUVs, respectively). Surprisingly, in contrast to the behavior in living cells, all peptides readily entered the giant vesicles ( > 1 μm) as proved by confocal microscopy, while none of them could cross the membranes of LUVs (100 nm). For determination of the location of the peptides in the LUVs, a new concept was introduced, based on sensitive resonance energy transfer (RET) measurements of the enhanced fluorescence of acceptor fluorophores present solely in the inner leaflet. An easily adopted method to prepare such asymmetrically labeled liposomes is described. The membrane insertion depths of the tryptophan moieties of the peptides were determined by use of brominated lipids and found to be very similar for all of the peptides studied. We also demonstrate that infrared spectroscopy on the lipid carbonyl stretch vibration peak is a convenient technique to determine phospholipid concentration. © 2004 Elsevier B.V. All rights reserved.
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| 52. |
- Quesada, E., et al.
(author)
-
Synthesis and fluorescence properties of novel transmembrane probes and determination of their orientation within vesicles
- 2000
-
In: Helvetica Chimica Acta. - 1522-2675 .- 0018-019X. ; 83:9, s. 2464-2476
-
Journal article (peer-reviewed)abstract
- Two novel transmembrane fluorescent diester probes D and E bearing an anthracenediyl moiety in the middle of the molecule have been synthesized. Their absorption and fluorescence spectra in CHCl3 solution as well as their fluorescence characteristics in dimyristoylphosphatidylcholine (DMPC) large unilamellar vesicles were determined. Although their absorption spectra (first transition, S-0 --> S-1) present a good overlap with the fluorescence spectrum of tryptophan, only probe E could be a good acceptor for the energy-transfer experiments, since a strong overlap exists between the absorption spectrum of tryptophan and the second transition (S-0 --> S-2) of the absorption spectrum of probe D. The Forster critical distance R-0 for energy transfer between tryptophan (donor) and probe E (acceptor) is found to be 23-24 Angstrom. Finally, linear-dichroism studies on shear-deformed DMPC vesicles show the incorporated probe E to lie essentially perpendicular to the bilayer plane. These results establish that probe E could be useful in the study of membrane-bound protein topography by the fluorescence-energy-transfer method.
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| 53. |
- Ratilainen, Tommi, 1970, et al.
(author)
-
A simple model for gene targeting
- 2001
-
In: Biophysical Journal. - 0006-3495 .- 1542-0086. ; 81:5, s. 2876-2885
-
Journal article (peer-reviewed)abstract
- Sequence-specific binding to genomic-size DNA sequences by artificial agents is of major interest for the development of gene-targeting strategies, gene-diagnostic applications, and biotechnical tools. The binding of one such agent, peptide nucleic acid (PNA), to a randomized human genome has been modeled with statistical mass action calculations. With the length of the PNA probe, the average per-base binding constant k(0), and the binding affinity loss of a mismatched base pair as main parameters, the specificity was gauged as a "therapeutic ratio" G = maximum safe [PNA](tot)/minimal efficient [PNA](tot). This general, though simple, model suggests that, above a certain threshold length of the PNA, the microscopic binding constant k(0) is the primary determinant for optimal discrimination, and that only a narrow range of rather low k(0) values gives a high therapeutic ratio G. For diagnostic purposes, the value of k(0) could readily be modulated by changing the temperature, due to the substantial DeltaH(o) associated with the binding equilibrium. Applied to gene therapy, our results stress the need for appropriate control of the binding constant and added amount of the gene-targeting agent, to meet the varying conditions (ionic strength, presence of competing DNA-binding molecules) found in the cell.
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| 54. |
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| 55. |
- Ratilainen, Tommi, 1970, et al.
(author)
-
Thermodynamics of sequence-specific binding of PNA to DNA
- 2000
-
In: Biochemistry. - : American Chemical Society (ACS). - 1520-4995 .- 0006-2960. ; 39:26, s. 7781-7791
-
Journal article (peer-reviewed)abstract
- For further characterization of the hybridization properties of peptide nucleic acids (PNAs), the thermodynamics of hybridization of mixed sequence PNA-DNA duplexes have been studied. We have characterized the binding of PNA to DNA in terms of binding affinity (perfectly matched duplexes) and sequence specificity of binding (singly mismatched duplexes) using mainly absorption hypochromicity melting curves and isothermal titration calorimetry. For perfectly sequence-matched duplexes of varying lengths (6-20 bp), the average free energy of binding (Delta G degrees) was determined to be -6.5 +/- 0.3 kT mol(-1) bp(-1), corresponding to a microscopic binding constant of about 14 M-1 bp(-1). A variety of single mismatches were introduced in 9- and 12-mer PNA-DNA duplexes. Melting temperatures (T-m) of 9- and 12-mer PNA-DNA duplexes with a single mismatch dropped typically 15-20 degrees C relative to that of the perfectly matched sequence with a corresponding free energy penalty of about 15 kT mol(-1) bp(-1). The average cost of a single mismatch is therefore estimated to be on the order of or larger than the gain of two matched base pairs, resulting in an apparent binding constant of only 0.02 M-1 per mismatch. The impact of a mismatch was found to be dependent on the neighboring base pairs. To a first approximation, increasing the stability of the surrounding region, i.e., the distribution of A.T and G.C base pairs, decreases the effect of the introduced mismatch.
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| 56. |
- Ray, Arghya, et al.
(author)
-
Peptide nucleic acid (PNA): its medical and biotechnical applications and promise for the future
- 2000
-
In: FASEB Journal. - 1530-6860 .- 0892-6638. ; 14:9, s. 1041-1060
-
Journal article (peer-reviewed)abstract
- Synthetic molecules that can bind with high sequence specificity to a chosen target in a gene sequence are of major interest in medicinal and biotechnological contexts. They show promise for the development of gene therapeutic agents, diagnostic devices for genetic analysis, and as molecular tools for nucleic acid manipulations. Peptide nucleic acid (PNA) is a nucleic acid analog in which the sugar phosphate backbone of natural nucleic acid has been replaced by a synthetic peptide backbone usually formed from N-(2-amino-ethyl)-glycine units, resulting in an achiral and uncharged mimic. It is chemically stable and resistant to hydrolytic (enzymatic) cleavage and thus not expected to be degraded inside a living cell. PNA is capable of sequence-specific recognition of DNA and RNA obeying the Watson-Crick hydrogen bonding scheme, and the hybrid complexes exhibit extraordinary thermal stability and unique ionic strength effects. It may also recognize duplex homopurine sequences of DNA to which it binds by strand invasion, forming a stable PNA-DNA-PNA tripler with a looped-out DNA strand. Since its discovery, PNA has attracted major attention at the interface of chemistry and biology because of its interesting chemical, physical, and biological properties and its potential to act as an active component for diagnostic as well as pharmaceutical applications. In vitro studies indicate that PNA could inhibit both transcription and translation of genes to which it has been targeted, which holds promise for its use for antigene and antisense therapy. However, as with other high molecular mass drugs, the delivery of PNA, involving passage through the cell membrane, appears to be a general problem.-Ray, A., Norden, B. Peptide nucleic acid (PNA): its medical and biotechnical applications and promise for the future.
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| 57. |
- Reymer, Anna, 1983, et al.
(author)
-
Structure of human Rad51 protein filament from molecular modeling and site-specific linear dichroism spectroscopy
- 2009
-
In: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 106:32, s. 13248-13253
-
Journal article (peer-reviewed)abstract
- To get mechanistic insight into the DNA strand-exchange reaction of homologous recombination, we solved a filament structure of a human Rad51 protein, combining molecular modeling with experimental data. We build our structure on reported structures for central and N-terminal parts of pure (uncomplexed) Rad51 protein by aid of linear dichroism spectroscopy, providing angular orientations of substituted tyrosine residues of Rad51-dsDNA filaments in solution. The structure, validated by comparison with an electron microscopy density map and results from mutation analysis, is proposed to represent an active solution structure of the nucleo-protein complex. An inhomogeneously stretched double-stranded DNA fitted into the filament emphasizes the strategic positioning of 2 putative DNA-binding loops in a way that allows us speculate about their possibly distinct roles in nucleo-protein filament assembly and DNA strand-exchange reaction. The model suggests that the extension of a single-stranded DNA molecule upon binding of Rad51 is ensured by intercalation of Tyr-232 of the L1 loop, which might act as a docking tool, aligning protein monomers along the DNA strand upon filament assembly. Arg-235, also sitting on L1, is in the right position to make electrostatic contact with the phosphate backbone of the other DNA strand. The L2 loop position and its more ordered compact conformation makes us propose that this loop has another role, as a binding site for an incoming double-stranded DNA. Our filament structure and spectroscopic approach open the possibility of analyzing details along the multistep path of the strand-exchange reaction.
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| 58. |
- Rodger, A., et al.
(author)
-
DNA as a Catalyst and Catalytic Template for the Racemisation of Metal Tris-Phenanthroline Complexes
- 2002
-
In: European Journal of Inorganic Chemistry. - 1434-1948 .- 1099-0682. ; :1, s. 49-53
-
Journal article (peer-reviewed)abstract
- [Fe(1,10-phenanthroline)(3)](2+) is known to racemise in solution and DNA is known to shift the equilibrium point of the Delta/Lambda[Fe(1,10-phenanthroline)(3)](2+) mixture from 50:50 to a position favouring a slight excess of the Delta enantiomer. In this paper it is shown that DNA catalyses the racemisation reaction, presumably by providing vibrational energy to species that bind in a less favoured mode for that enantiomer. The racemisation of [Co(1,10-phenanthroline)(3)](3+), however, is not enhanced by DNA unless [Co(1,10-phenanthroline)(3)](2+) is also added to the solution. In this case, the DNA acts as a template to facilitate the Co-II reversible arrow Co-III electron transfer. Racemisation occurs while the [Co(1,10-phenanthroline)(3)](n+) is in the labile Co-II form.
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| 59. |
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| 60. |
- Sandin, Peter, 1977, et al.
(author)
-
Thermodynamic Aspects of DNA Nanoconstruct Stability and Design
- 2009
-
In: Journal of Physical Chemistry C. - : American Chemical Society (ACS). - 1932-7447 .- 1932-7455. ; 113:15, s. 5941-5946
-
Journal article (peer-reviewed)abstract
- In the present study, we use the fluorescent DNA base analog tCå to investigate the thermal stability of a small DNA hexagon and the thermodynamie factors that govern the formation of such a structure. The DNA molecule is becoming increasingly popular as a material for bottom-up construction of nanostructures; however, relatively little attention has been given to the thermodynamics of such biomacromolecule-based constructs. With the goal of increasing information density and structural complexity, the size of the nanoarchitectures decreases and, more importantly, the fine structure is becoming more detailed. In this process the thermal stability and formation of unwanted byproducts will become critical features to consider in the design and assembly of such structures. Using tCå as a fluorescent probe in fluorescence monitored DNA melting allows for individually observing the denaturing of each of the six 10-mer sides in the pseudohexagonal multicomponent system. Experimental results demonstrate that the ring-opening of the cyclized hexamer is virtually exclusive to one side and that the stability of this side is increased as a result of the cyclization. Moreover, a theoretical model describing the formation and melting of the nanostructure is presented. The results show that the cyclized structure is thermodynamically favored over linear polymeric structures under the conditions and concentrations used for the self-assembly. © 2009 American Chemical Society.
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