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| 2. |
- Füchtbauer, Anders Foller, 1984, et al.
(författare)
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Fluorescent RNA cytosine analogue - an internal probe for detailed structure and dynamics investigations
- 2017
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- The bright fluorescent cytosine analogue tCO stands out among fluorescent bases due to its virtually unquenched fluorescence emission in duplex DNA. However, like most reported base analogues, it has not been thoroughly characterized in RNA. We here report on the first synthesis and RNA-incorporation of tCO, and characterize its base-mimicking and fluorescence properties in RNA. As in DNA, we find a high quantum yield inside RNA duplexes (< Phi(F)> = 0.22) that is virtually unaffected by the neighbouring bases (Phi(F) = 0.20-=0.25), resulting in an average brightness of 1900 M-1 cm(-1). The average fluorescence lifetime in RNA duplexes is 4.3 ns and generally two lifetimes are required to fit the exponential decays. Fluorescence properties in ssRNA are defined by a small increase in average quantum yield (< Phi(F) > = 0.24) compared to dsRNA, with a broader distribution (Phi(F) = 0.17-=0.34) and slightly shorter average lifetimes. Using circular dichroism, we find that the tCO-modified RNA duplexes form regular A-form helices and in UV-melting experiments the stability of the duplexes is only slightly higher than that of the corresponding natural RNA ( = + 2.3 degrees C). These properties make tC(0) a highly interesting fluorescent RNA base analogue for detailed FRET-based structural measurements, as a bright internal label in microscopy, and for fluorescence anisotropy measurements of RNA dynamics.
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| 3. |
- Preus, S., et al.
(författare)
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The photoinduced transformation of fluorescent DNA base analogue tC triggers DNA melting
- 2013
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Ingår i: Photochemical and Photobiological Sciences. - : Springer Science and Business Media LLC. - 1474-9092 .- 1474-905X. ; 12:8, s. 1416-1422
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Tidskriftsartikel (refereegranskat)abstract
- While fluorescent analogues of the canonical nucleobases have proven to be highly valuable in a large number of applications, up until today, fluorescent DNA base analogues remain virtually inapplicable for single-molecule fluorescence experiments which require extremely bright and photostable dyes. Insight into the photodegradation processes of these fluorophores is thus a key step in the continuous development towards dyes with improved performances. Here, we show that the commercially available fluorescent nucleobase analogue tC under intense long-term illumination and in the presence of O-2 is degraded to form a single photoreaction product which we suggest to be the sulfoxide form of tC. The photoproduct is characterized by a blue-shifted absorption and a less intense fluorescence compared to that of tC. Interestingly, when tC is positioned inside double-stranded DNA this photodriven conversion of tC to its photoproduct greatly reduces the duplex stability of the overall double helix in which the probe is positioned. Since tC can be excited selectively at 400 nm, well outside the absorption band of the natural DNA bases, this observation points towards the application of tC as a general light-triggered switch of DNA duplex stability.
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| 4. |
- Börjesson, Karl, 1982, et al.
(författare)
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Nucleic Acid Base Analog FRET-Pair Facilitating Detailed Structural Measurements in Nucleic Acid Containing Systems
- 2009
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 131:12, s. 4288-4293
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Tidskriftsartikel (refereegranskat)abstract
- We present the first nucleobase analog fluorescence resonance energy transfer (FRET)-pair. The pair consists of tC O , 1,3-diaza-2- oxophenoxazine, as an energy donor and the newly developed tC nitro , 7-nitro-1,3-diaza-2-oxophenothiazine, as an energy acceptor. The FRET-pair successfully monitors distances covering up to more than one turn of the DNA duplex. Importantly, we show that the rigid stacking of the two base analogs, and consequently excellent control of their exact positions and orientations, results in a high control of the orientation factor and hence very distinct FRET changes as the number of bases separating tC O and tCnitro is varied. A set of DNA strands containing the FRET-pair at wisely chosen locations will, thus, make it possible to accurately distinguish distance- from orientation-changes using FRET. In combination with the good nucleobase analog properties, this points toward detailed studies of the inherent dynamics of nucleic acid structures. Moreover, the placement of FRET-pair chromophores inside the base stack will be a great advantage in studies where other (biomacro)molecules interact with the nucleic acid. Lastly, our study gives possibly the first truly solid experimental support to the dependence of energy transfer efficiency on orientation of involved transition dipoles as predicted by the Forster theory. © 2009 American Chemical Society.
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| 5. |
- Dierckx, Anke, 1986, et al.
(författare)
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Quadracyclic Adenine: A Non-Perturbing Fluorescent Adenine Analogue
- 2012
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Ingår i: Chemistry - A European Journal. - : Wiley. - 1521-3765 .- 0947-6539. ; 18:19, s. 5987-5997
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Tidskriftsartikel (refereegranskat)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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| 6. |
- Dumat, Blaise, 1984, et al.
(författare)
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Second-Generation Fluorescent Quadracyclic Adenine Analogues: Environment-Responsive Probes with Enhanced Brightness
- 2015
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Ingår i: Chemistry - A European Journal. - : Wiley. - 0947-6539 .- 1521-3765. ; 21:10, s. 4039-4048
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Tidskriftsartikel (refereegranskat)abstract
- Fluorescent base analogues comprise a group of increasingly important molecules for the investigation of nucleic acid structure, dynamics, and interactions with other molecules. Herein, we report on the quantum chemical calculation aided design, synthesis, and characterization of four new putative quadracyclic adenine analogues. The compounds were efficiently synthesized from a common intermediate through a two-step pathway with the Suzuki-Miyaura coupling as the key step. Two of the compounds, qAN1 and qAN4, display brightnesses (εΦF) of 1700 and 2300, respectively, in water and behave as wavelength-ratiometric pH probes under acidic conditions. The other two, qAN2 and qAN3, display lower brightnesses but exhibit polarity-sensitive dual-band emissions that could prove useful to investigate DNA structural changes induced by DNA-protein or -drug interactions. The four qANs are very promising microenvironment-sensitive fluorescent adenine analogues that display considerable brightness for such compounds. Polarity and pH probes: 2-Aminopurine has long been the standard for fluorescent base analogues. Four new fluorescent probes suitable for the replacement of adenine in nucleic acids are presented. Based on their high structural similarity to their parent compound, quadracyclic adenine, they have the potential to be excellent A analogues. Their improved photophysical properties also suggest that they could be significantly brighter than 2-aminopurine inside nucleic acid systems (see figure; ΦF: fluorescence quantum yield).
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| 7. |
- Larsen, Anders Foller, 1984, et al.
(författare)
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Development of bright fluorescent quadracyclic adenine analogues: TDDFT-calculation supported rational design
- 2015
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 5
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Tidskriftsartikel (refereegranskat)abstract
- Fluorescent base analogues (FBAs) comprise a family of increasingly important molecules for the investigation of nucleic acid structure and dynamics. We recently reported the quantum chemical calculation supported development of four microenvironment sensitive analogues of the quadracyclic adenine (qA) scaffold, the qANs, with highly promising absorptive and fluorescence properties that were very well predicted by TDDFT calculations. Herein, we report on the efficient synthesis, experimental and theoretical characterization of nine novel quadracyclic adenine derivatives. The brightest derivative, 2-CNqA, displays a 13-fold increased brightness (epsilon Phi(F) = 4500) compared with the parent compound qA and has the additional benefit of being a virtually microenvironment-insensitive fluorophore, making it a suitable candidate for nucleic acid incorporation and use in quantitative FRET and anisotropy experiments. TDDFT calculations, conducted on the nine novel qAs a posteriori, successfully describe the relative fluorescence quantum yield and brightness of all qA derivatives. This observation suggests that the TDDFT-based rational design strategy may be employed for the development of bright fluorophores built up from a common scaffold to reduce the otherwise costly and time-consuming screening process usually required to obtain useful and bright FBAs.
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| 8. |
- Preus, S., et al.
(författare)
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Advances in Quantitative FRET-Based Methods for Studying Nucleic Acids
- 2012
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Ingår i: ChemBioChem. - : Wiley. - 1439-7633 .- 1439-4227. ; 13:14, s. 1990-2001
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Tidskriftsartikel (refereegranskat)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.
(författare)
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Characterization of Nucleobase Analogue FRET Acceptor tC(nitro)
- 2010
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Ingår i: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-5207 .- 1520-6106. ; 114:2, s. 1050-1056
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Tidskriftsartikel (refereegranskat)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.
(författare)
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FRETmatrix: a general methodology for the simulation and analysis of FRET in nucleic acids
- 2013
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 41:1, s. Article Number: e18-
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Tidskriftsartikel (refereegranskat)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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