| 1. |
- Dierckx, Anke, 1986, et al.
(författare)
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Characterization of photophysical and base-mimicking properties of a novel fluorescent adenine analogue in DNA
- 2011
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 39:10, s. 4513-4524
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Tidskriftsartikel (refereegranskat)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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| 2. |
- Dierckx, Anke, 1986
(författare)
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Expanding the alphabet of fluorescent nucleic acid base analogues: Characterization of new members
- 2011
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Fluorescence has proven to be a powerful tool for studying structure, dynamics and interactions of biomolecules such as proteins, RNA and DNA. However, since the natural DNA/RNA nucleobases are virtually non-fluorescent, there is a need for developing fluorescent nucleic acid markers. Many dyes have been designed to be covalently attached by linkers as external probes to the DNA. Nevertheless, there are some important limitations with these markers, such as perturbations caused to the duplex structure and the lack of site-specific information. Therefore there is an increasing focus on the development of fluorescent nucleic acid base analogues (FBAs). These are artificial DNA bases, which are significantly fluorescent, resemble the structure of one of the natural nucleobases, have some hydrogen bonding capacity to the opposite base and preserve the B-DNA helical structure. In this thesis the photophysical and base-mimicking properties of two new fluorescent adenine analogues, triazole adenine (AT) and quadracyclic adenine (qA) are presented. Both analogues proved to be very promising compared to the widely used commercially available 2-aminopurine (2-AP). AT shows a high fluorescence both as a monomer and in DNA. Quantum yields in single-strands and duplexes reach values up to ten and five times higher, respectively, than maximum values reported for 2-AP. Furthermore, as for 2-AP and most other FBAs, AT causes minor perturbations to the B-DNA duplex structure. Interestingly, we found AT to be capable of forming base-pairs both with thymine and adenine. The second fluorescent adenine analogue discussed in this work, qA, is moderately fluorescent as a monomer. However, inside single-and double-stranded DNA, quantum yields reach values which are four times higher or comparable, to corresponding values for 2-AP. Importantly, qA causes no perturbations to the DNA duplex and even stabilizes it depending on the surrounding sequence. Furthermore, in contrast to 2-AP, qA shows specific base-pairing with thymine. These properties of qA are unprecedented for fluorescent adenine analogues. The above mentioned qualities make AT and qA attractive alternatives for 2-AP in future applications. Finally, these two new fluorescent adenine analogues may help to form a better understanding of the relationship between the structural and fluorescence properties of FBAs.
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| 3. |
- Dierckx, Anke, 1986
(författare)
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Fluorescent Nucleobase Analogues and their use for Investigating DNA Interactions
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Ever since unravelling the structure of DNA, an expanding research field has emerged with ongoing efforts dedicated to increase our understanding of the molecule of life. Since the natural nucleobases are virtually non-emissive, it has been a challenge for decades to ‘light up’ DNA/RNA in order to investigate their properties utilizing fluorescence techniques. This thesis focuses on fluorescent nucleobase analogues (FBAs) as probes for fluorescently labeling DNA and investigating its interactions, for example, with proteins. These artificial nucleobases attempt to closely mimic the characteristics of natural bases, while introducing fluorescence properties to the system. The first part of this work comprises the characterization of both new and established FBAs. Photophysical and base-mimicking properties of two fluorescent adenine analogues, triazole adenine (AT) and quadracyclic adenine (qA) are presented. Both exhibit promising features compared to the widely used commercially available adenine mimic 2-aminopurine (2-AP). Even though AT shows promising emission as a monomer and in certain DNA surroundings, it destabilizes the B-DNA duplex structure, most likely due to its C8-triazole extension. In order to overcome this effect, a new family of triazole adenine analogues extended on the 7-position was synthesized and photophysically characterized. The second thoroughly characterized adenine analogue, qA, is moderately fluorescent both as a monomer and inside DNA but in contrast to AT, the two-ring extension on qA is suggested to be well accommodated in the major groove and renders the DNA-duplex unperturbed or even stabilized, depending on the surrounding sequence. Finally, the photostability of tC, an already established FBA of the tricyclic cytosine family, was investigated. The latter yields a single photoproduct with a decreased fluorescence, which destabilizes DNA duplexes.In the second part of this work, the tricyclic cytosine FBA FRET-pair, tCO-tCnitro was applied in exploring the role of the mammalian transcription factor A in mitochondrial transcription. Furthermore, it was called upon to help resolve the order of events in which the different components of the transcription machinery initiate transcription.
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| 4. |
- 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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| 5. |
- Lawson, Christopher, 1968, et al.
(författare)
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Synthesis and photophysical characterisation of new fluorescent triazole adenine analogues
- 2014
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Ingår i: Organic & Biomolecular Chemistry. - : Royal Society of Chemistry (RSC). - 1477-0520 .- 1477-0539. ; 12:28, s. 5158-5167
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Tidskriftsartikel (refereegranskat)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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| 6. |
- 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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| 7. |
- Shi, Yonghong, et al.
(författare)
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Mammalian transcription factor A is a core component of the mitochondrial transcription machinery
- 2012
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Ingår i: Proceedings of the National Academy of Science of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 109:41, s. 16510-16515
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Tidskriftsartikel (refereegranskat)abstract
- Transcription factor A (TFAM) functions as a DNA packaging factor in mammalian mitochondria. TFAM also binds sequence-specifically to sites immediately upstream of mitochondrial promoters, but there are conflicting data regarding its role as a core component of the mitochondrial transcription machinery. We here demonstrate that TFAM is required for transcription in mitochondrial extracts as well as in a reconstituted in vitro transcription system. The absolute requirement of TFAM can be relaxed by conditions that allow DNA breathing, i.e., low salt concentrations or negatively supercoiled DNA templates. The situation is thus very similar to that described in nuclear RNA polymerase II-dependent transcription, in which the free energy of supercoiling can circumvent the need for a subset of basal transcription factors at specific promoters. In agreement with these observations, we demonstrate that TFAM has the capacity to induce negative supercoils in DNA, and, using the recently developed nucleobase analog FRET-pair tC(O)-tC(nitro), we find that TFAM distorts significantly the DNA structure. Our findings differ from recent observations reporting that TFAM is not a core component of the mitochondrial transcription machinery. Instead, our findings support a model in which TFAM is absolutely required to recruit the transcription machinery during initiation of transcription.
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