| 1. |
- Carlsson, Nils, 1978, et al.
(author)
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DNA hosted and aligned in aqueous interstitia of a lamellar liquid crystal - a membrane-biomacromolecule interaction model system
- 2013
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In: Soft Matter. - : Royal Society of Chemistry (RSC). - 1744-6848 .- 1744-683X. ; 9:33, s. 7951-7959
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Journal article (peer-reviewed)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.
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| 2. |
- Dierckx, Anke, 1986, et al.
(author)
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Characterization of photophysical and base-mimicking properties of a novel fluorescent adenine analogue in DNA
- 2011
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In: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 39:10, s. 4513-4524
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Journal article (peer-reviewed)abstract
- To increase the diversity of fluorescent base analogues with improved properties, we here present the straightforward click-chemistry-based synthesis of a novel fluorescent adenine-analogue triazole adenine (A(T)) and its photophysical characterization inside DNA. A(T) shows promising properties compared to the widely used adenine analogue 2-aminopurine. Quantum yields reach > 20% and > 5% in single- and double-stranded DNA, respectively, and show dependence on neighbouring bases. Moreover, A(T) shows only a minor destabilization of DNA duplexes, comparable to 2-aminopurine, and circular dichroism investigations suggest that A(T) only causes minimal structural perturbations to normal B-DNA. Furthermore, we find that A(T) shows favourable base-pairing properties with thymine and more surprisingly also with normal adenine. In conclusion, A(T) shows strong potential as a new fluorescent adenine analogue for monitoring changes within its microenvironment in DNA.
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| 3. |
- Dierckx, Anke, 1986, et al.
(author)
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Quadracyclic Adenine: A Non-Perturbing Fluorescent Adenine Analogue
- 2012
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In: Chemistry - A European Journal. - : Wiley. - 1521-3765 .- 0947-6539. ; 18:19, s. 5987-5997
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Journal article (peer-reviewed)abstract
- Fluorescent-base analogues (FBAs) comprise a group of increasingly important molecules for the investigation of nucleic acid structure and dynamics as well as of interactions between nucleic acids and other molecules. Here, we report on the synthesis, detailed spectroscopic characterisation and base-pairing properties of a new environment-sensitive fluorescent adenine analogue, quadracyclic adenine (qA). After developing an efficient route of synthesis for the phosphoramidite of qA it was incorporated into DNA in high yield by using standard solid-phase synthesis procedures. In DNA qA serves as an adenine analogue that preserves the B-form and, in contrast to most currently available FBAs, maintains or even increases the stability of the duplex. We demonstrate that, unlike fluorescent adenine analogues, such as the most commonly used one, 2-aminopurine, and the recently developed triazole adenine, qA shows highly specific base-pairing with thymine. Moreover, qA has an absorption band outside the absorption of the natural nucleobases (>300 nm) and can thus be selectively excited. Upon excitation the qA monomer displays a fluorescence quantum yield of 6.8?% with an emission maximum at 456 nm. More importantly, upon incorporation into DNA the fluorescence of qA is significantly less quenched than most FBAs. This results in quantum yields that in some sequences reach values that are up to fourfold higher than maximum values reported for 2-aminopurine. To facilitate future utilisation of qA in biochemical and biophysical studies we investigated its fluorescence properties in greater detail and resolved its absorption band outside the DNA absorption region into distinct transition dipole moments. In conclusion, the unique combination of properties of qA make it a promising alternative to current fluorescent adenine analogues for future detailed studies of nucleic acid-containing systems.
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| 4. |
- Lawson, Christopher, 1968, et al.
(author)
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Synthesis and photophysical characterisation of new fluorescent triazole adenine analogues
- 2014
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In: Organic & Biomolecular Chemistry. - : Royal Society of Chemistry (RSC). - 1477-0520 .- 1477-0539. ; 12:28, s. 5158-5167
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Journal article (peer-reviewed)abstract
- Fluorescent nucleic acid base analogues are powerful probes of DNA structure. Here we describe the synthesis and photo-physical characterisation of a series of 2-(4-amino-5-(1H-1,2,3-triazol-4-yl)-7H-pyrrolo-[2,3-d] pyrimidin-7-yl) and 2-(4-amino-3-(1H-1,2,3-triazol-4-yl)-1H-pyrazolo[3,4-d] pyrimidin-1-yl) analogues via Sonogashira cross-coupling and [3 + 2]-cycloaddition reactions as the key steps in the synthesis. Compounds with a nitrogen atom in position 8 showed an approximately ten-fold increase in quantum yield and decreased Stokes shift compared to analogues with a carbon atom in position 8. Furthermore, the analogues containing nitrogen in the 8-position showed a more red-shifted and structured absorption as opposed to those which have a carbon incorporated in the same position. Compared to the previously characterised C8-triazole modified adenine, the emissive potential was significantly lower (tenfold or more) for this new family of triazoles-adenine compounds. However, three of the compounds have photophysical properties which will make them interesting to monitor inside DNA.
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| 5. |
- Lundberg, Erik, 1981, et al.
(author)
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A new fixation strategy for addressable nano-network building blocks
- 2010
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In: Chemical Communications. - 1364-548X .- 1359-7345. ; 46:21, s. 3714-3716
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Journal article (peer-reviewed)abstract
- Rapid controlled self-assembly makes DNA ideal for building nanostructures. A problem using hybridized intermediates in hierarchic assembly is their thermodynamic lability. We demonstrate a click-fixation technology by which robust hexagonal DNA modules can be made. This principle is applicable to a wide variety of DNA nanoconstructs.
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| 6. |
- Lundberg, Erik, 1981, et al.
(author)
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Controlling and Monitoring Orientation of DNA Nanoconstructs on Lipid Surfaces
- 2013
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In: Langmuir. - : American Chemical Society (ACS). - 1520-5827 .- 0743-7463. ; 29:1, s. 285-293
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Journal article (peer-reviewed)abstract
- Its extraordinary self-assembly property, with potential to form nonperiodic structures with unique addressability, makes DNA ideal for fabrication of advanced nanostructures. We here demonstrate the controllable tethering of a hexagonal DNA nanostructure in two distinct orientations at the lipid bilayer of a liposome functioning as a soft-matter support. With polarized light (linear dichroism) applied to the flow-aligned liposomes, we show that the construct is preferentially in a parallel alignment with the lipid surface when two anchors are attached while with one anchor only a perpendicular orientation is observed.
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| 7. |
- Lundberg, Erik, 1981, et al.
(author)
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Nanofabrication Yields. Hybridization and Click-Fixation of Polycyclic DNA Nanoassemblies
- 2011
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In: ACS Nano. - : American Chemical Society (ACS). - 1936-086X .- 1936-0851. ; 5:9, s. 7565-7575
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Journal article (peer-reviewed)abstract
- We demonstrate the stepwise assembly of a fully addressable polycyclic DNA hexagon nanonetwork for the preparation of a lour-ring system, one of the biggest networks yet constructed from tripodal building blocks. We find that the yield exhibits a distinct upper level
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| 8. |
- Preus, S., et al.
(author)
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Advances in Quantitative FRET-Based Methods for Studying Nucleic Acids
- 2012
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In: ChemBioChem. - : Wiley. - 1439-7633 .- 1439-4227. ; 13:14, s. 1990-2001
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Journal article (peer-reviewed)abstract
- Förster resonance energy transfer (FRET) is a powerful tool for monitoring molecular distances and interactions at the nanoscale level. The strong dependence of transfer efficiency on probe separation makes FRET perfectly suited for on/off experiments. To use FRET to obtain quantitative distances and three-dimensional structures, however, is more challenging. This review summarises recent studies and technological advances that have improved FRET as a quantitative molecular ruler in nucleic acid systems, both at the ensemble and at the single-molecule levels.
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| 9. |
- Preus, S., et al.
(author)
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Characterization of Nucleobase Analogue FRET Acceptor tC(nitro)
- 2010
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In: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-5207 .- 1520-6106. ; 114:2, s. 1050-1056
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Journal article (peer-reviewed)abstract
- The fluorescent nucleobase analogues of the tricyclic cytosine (tC) family, tC and tC(O), possess high fluorescence quantum yields and single fluorescence lifetimes, even after incorporation into double-stranded DNA, which make these base analogues particularly useful as fluorescence resonance energy transfer (FRET) probes. Recently, we reported the first all-nucleobase FRET pair consisting of tC(O) as the donor and the novel tC(nitro) as the acceptor. The rigid and well-defined position of this FRET pair inside the DNA double helix, and consequently excellent control of the orientation factor in the FRET efficiency, are very promising features for future studies of nucleic acid structures. Here, we provide the necessary spectroscopic and photophysical characterization Of tC(nitro) needed in order to utilize this probe as a FRET acceptor in nucleic acids. The lowest energy absorption band from 375 to 525 nm is shown to be the result of a single in-plane polarized electronic transition oriented similar to 27 degrees from the molecular long axis, This band overlaps the emission bands of both tC and tC(O), and the Forster characteristics of these donor-acceptor pairs are calculated for double-stranded DNA scenarios. In addition, the UV-vis absorption of tC(nitro) is monitored in a broad pH range and the neutral form is found to be totally predominant under physiological conditions with a pK(a) of 11.1. The structure and electronic spectrum Of tC(nitro) is further characterized by density functional theory calculations.
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| 10. |
- Preus, S., et al.
(author)
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FRETmatrix: a general methodology for the simulation and analysis of FRET in nucleic acids
- 2013
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In: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 41:1, s. Article Number: e18-
-
Journal article (peer-reviewed)abstract
- Forster resonance energy transfer (FRET) is a technique commonly used to unravel the structure and conformational changes of biomolecules being vital for all living organisms. Typically, FRET is performed using dyes attached externally to nucleic acids through a linker that complicates quantitative interpretation of experiments because of dye diffusion and reorientation. Here, we report a versatile, general methodology for the simulation and analysis of FRET in nucleic acids, and demonstrate its particular power for modelling FRET between probes possessing limited diffusional and rotational freedom, such as our recently developed nucleobase analogue FRET pairs (base-base FRET). These probes are positioned inside the DNA/RNA structures as a replacement for one of the natural bases, thus, providing unique control of their position and orientation and the advantage of reporting from inside sites of interest. In demonstration studies, not requiring molecular dynamics modelling, we obtain previously inaccessible insight into the orientation and nanosecond dynamics of the bases inside double-stranded DNA, and we reconstruct high resolution 3D structures of kinked DNA. The reported methodology is accompanied by a freely available software package, FRETmatrix, for the design and analysis of FRET in nucleic acid containing systems.
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