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- 2019
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Journal article (peer-reviewed)
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| 2. |
- Bood, Mattias, et al.
(author)
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Pentacyclic adenine: a versatile and exceptionally bright fluorescent DNA base analogue
- 2018
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In: Chemical Science. - : Royal Society of Chemistry (RSC). - 2041-6520 .- 2041-6539. ; 9:14, s. 3494-3502
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Journal article (peer-reviewed)abstract
- Emissive base analogs are powerful tools for probing nucleic acids at the molecular level. Herein we describe the development and thorough characterization of pentacyclic adenine (pA), a versatile base analog with exceptional fluorescence properties. When incorporated into DNA, pA pairs selectively with thymine without perturbing the B-form structure and is among the brightest nucleobase analogs reported so far. Together with the recently established base analog acceptor qAnitro, pA allows accurate distance and orientation determination via Forster resonance energy transfer (FRET) measurements. The high brightness at emission wavelengths above 400 nm also makes it suitable for fluorescence microscopy, as demonstrated by imaging of single liposomal constructs coated with cholesterolanchored pA-dsDNA, using total internal reflection fluorescence microscopy. Finally, pA is also highly promising for two-photon excitation at 780 nm, with a brightness (5.3 GM) that is unprecedented for a base analog.
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| 3. |
- Füchtbauer, Anders Foller, 1984, et al.
(author)
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Lighting Up DNA with the Environment-Sensitive Bright Adenine Analogue qAN4
- 2020
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In: Chempluschem. - : Wiley. - 2192-6506. ; 85:2, s. 319-326
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Journal article (peer-reviewed)abstract
- The fluorescent adenine analogue qAN4 was recently shown to possess promising photophysical properties, including a high brightness as a monomer. Here we report the synthesis of the phosphoramidite of qAN4 and its successful incorporation into DNA oligonucleotides using standard solid-phase synthesis. Circular dichroism and thermal melting studies indicate that the qAN4-modification has a stabilizing effect on the B-form of DNA. Moreover, qAN4 base-pairs selectively with thymine with mismatch penalties similar to those of mismatches of adenine. The low energy absorption band of qAN4 inside DNA has its peak around 358 nm and the emission in duplex DNA is partly quenched and blue-shifted (ca. 410 nm), compared to the monomeric form. The spectral properties of the fluorophore also show sensitivity to pH; a property that may find biological applications. Quantum yields in single-stranded DNA range from 1-29 % and in duplex DNA from 1-7 %. In combination with the absorptive properties, this gives an average brightness inside duplex DNA of 275 M-1 cm(-1), more than five times higher than the most used environment-sensitive fluorescent base analogue, 2-aminopurine. Finally, we show that qAN4 can be used to advantage as a donor for interbase FRET applications in combination with adenine analogue qA(nitro) as an acceptor.
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| 4. |
- Lawson, Christopher, 1968, et al.
(author)
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Synthesis, oligonucleotide incorporation and fluorescence properties in DNA of a bicyclic thymine analogue
- 2018
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In: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 8
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Journal article (peer-reviewed)abstract
- Fluorescent base analogues (FBAs) have emerged as a powerful class of molecular reporters of location and environment for nucleic acids. In our overall mission to develop bright and useful FBAs for all natural nucleobases, herein we describe the synthesis and thorough characterization of bicyclic thymidine (bT), both as a monomer and when incorporated into DNA. We have developed a robust synthetic route for the preparation of the bT DNA monomer and the corresponding protected phosphoramidite for solid-phase DNA synthesis. The bT deoxyribonucleoside has a brightness value of 790 M(-1)cm(-1) in water, which is comparable or higher than most fluorescent thymine analogues reported. When incorporated into DNA, bT pairs selectively with adenine without perturbing the B-form structure, keeping the melting thermodynamics of the B-form duplex DNA virtually unchanged. As for most fluorescent base analogues, the emission of bT is reduced inside DNA (4.5- and 13-fold in single- and double-stranded DNA, respectively). Overall, these properties make bT an interesting thymine analogue for studying DNA and an excellent starting point for the development of brighter bT derivatives.
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| 5. |
- Sandberg Wranne, Moa, 1986, et al.
(author)
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Toward Complete Sequence Flexibility of Nucleic Acid Base Analogue FRET
- 2017
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In: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 139:27, s. 9271-9280
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Journal article (peer-reviewed)abstract
- Forster resonance energy transfer (FRET) using fluorescent base analogues is a powerful means of obtaining high-resolution nucleic acid structure and dynamics information that favorably complements techniques such as NMR and X-ray crystallography. Here, we expand the base base FRET repertoire with an adenine analogue FRET-pair. Phosphoramidite-protected quadracyclic 2'-deoxyadenosine analogues qAN1 (donor) and qA(nitro) (acceptor) were synthesized and incorporated into DNA by a genetic, reliable, and high-yielding route, and both constitute excellent adenine analogues. The donor, qAN1, has quantum yields reaching 21% and 11% in single- and double-strands, respectively. To the best of our knowledge, this results in the highest average brightness of an adenine analogue inside DNA. Its potent emissive features overlap well with the absorption of qA(nitro), and thus enable accurate FRET-measurements over more than one turn of B-DNA. As we have shown previously for our cytosine analogue FRET-pair, FRET between qAN1 and qA(nitro) positioned at different base separations inside DNA results in efficiencies that are highly dependent on both distance and orientation. This facilitates significantly enhanced resolution in FRET structure determinations, demonstrated here in a study of conformational changes of DNA upon binding of the minor groove binder netropsin. Finally, we note that the donor and acceptor, of our cytosine FRET-pair, tC degrees and tC(nitro), can be conveniently combined with the acceptor and donor of our current adenine pair, respectively. Consequently, our base analogues can now measure base base FRET between 3 of the 10 possible base combinations and, through base-complementarity, between all sequence positions in a duplex.
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| 6. |
- Wypijewska Del Nogal, Anna Wiktoria, 1982, et al.
(author)
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Getting DNA and RNA out of the dark with 2CNqA: a bright adenine analogue and interbase FRET donor
- 2020
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In: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 48:14, s. 7640-7652
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Journal article (peer-reviewed)abstract
- With the central role of nucleic acids there is a need for development of fluorophores that facilitate the visualization of processes involving nucleic acids without perturbing their natural properties and behaviour. Here, we incorporate a new analogue of adenine, 2CNqA, into both DNA and RNA, and evaluate its nucleobase-mimicking and internal fluorophore capacities. We find that 2CNqA displays excellent photophysical properties in both nucleic acids, is highly specific for thymine/uracil, and maintains and slightly stabilises the canonical conformations of DNA and RNA duplexes. Moreover, the 2CNqA fluorophore has a quantum yield in single-stranded and duplex DNA ranging from 10% to 44% and 22% to 32%, respectively, and a slightly lower one (average 12%) inside duplex RNA. In combination with a comparatively strong molar absorptivity for this class of compounds, the resulting brightness of 2CNqA inside double-stranded DNA is the highest reported for a fluorescent base analogue. The high, relatively sequence-independent quantum yield in duplexes makes 2CNqA promising as a nucleic acid label and as an interbase Forster resonance energy transfer (FRET) donor. Finally, we report its excellent spectral overlap with the interbase FRET acceptors qA(nitro) and tC(nitro), and demonstrate that these FRET pairs enable conformation studies of DNA and RNA.
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