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- Bood, Mattias, et al.
(författare)
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Pentacyclic adenine: a versatile and exceptionally bright fluorescent DNA base analogue
- 2018
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Ingår i: Chemical Science. - : Royal Society of Chemistry (RSC). - 2041-6520 .- 2041-6539. ; 9:14, s. 3494-3502
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Tidskriftsartikel (refereegranskat)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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| 2. |
- Bood, Mattias, et al.
(författare)
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Fluorescent nucleobase analogues for base-base FRET in nucleic acids: Synthesis, photophysics and applications
- 2018
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Ingår i: Beilstein Journal of Organic Chemistry. - : Beilstein Institut. - 1860-5397. ; 14, s. 114-129
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Tidskriftsartikel (refereegranskat)abstract
- Forster resonance energy transfer (FRET) between a donor nucleobase analogue and an acceptor nucleobase analogue, base-base FRET, works as a spectroscopic ruler and protractor. With their firm stacking and ability to replace the natural nucleic acid bases inside the base-stack, base analogue donor and acceptor molecules complement external fluorophores like the Cy-, Alexa- and ATTO-dyes and enable detailed investigations of structure and dynamics of nucleic acid containing systems. The first base-base FRET pair, tCO-tCnitro, has recently been complemented with among others the adenine analogue FRET pair, qAN1-qAnitro, increasing the flexibility of the methodology. Here we present the design, synthesis, photophysical characterization and use of such base analogues. They enable a higher control of the FRET orientation factor, κ2, have a different distance window of opportunity than external fluorophores, and, thus, have the potential to facilitate better structure resolution. Netropsin DNA binding and the B-to-Z-DNA transition are examples of structure investigations that recently have been performed using base.base FRET and that are described here. Base-base FRET has been around for less than a decade, only in 2017 expanded beyond one FRET pair, and represents a highly promising structure and dynamics methodology for the field of nucleic acids. Here we bring up its advantages as well as disadvantages and touch upon potential future applications. © 2018 Bood et al.
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| 3. |
- Lawson, Christopher, 1968, et al.
(författare)
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Synthesis, oligonucleotide incorporation and fluorescence properties in DNA of a bicyclic thymine analogue
- 2018
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 8
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Tidskriftsartikel (refereegranskat)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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| 4. |
- Sandberg Wranne, Moa, 1986
(författare)
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DNA and RNA base analogue FRET - from fluorophore design to biochemical applications
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis focuses on the development and use of fluorescent base analogues (FBAs). They are important tools in research concerning nucleic acids structure, dynamics and interactions. FBAs are fluorescent molecules that are structurally similar to the natural nucleobases and can therefore replace them inside nucleic acids without significantly perturbing the properties of the nucleic acid. The design of new FBAs is often challenging due to the limitations imposed on their structure by the overall structure of nucleic acids. This thesis starts by describing the development and characterization of a large set of new potential adenine analogues, all based on the structure of the FBA qA, and how TDDFT calculations were utilized to aid the design. Among these, three fluorescent (qAN1, qAN4 and pA) and one non-fluorescent (qAnitro) analogue have been incorporated and characterized inside DNA as well. They are all good adenine analogues, i.e . they do not perturb the structure or stability of DNA duplexes significantly. The three fluorescent analogues are all significantly brighter than the parent compound qA, and importantly, pA is the brightest adenine analogue inside DNA reported to date. The thesis also describes the development and characterization of a good thymine analogue, bT, which might serve as the starting point for development of brighter thymine analogues, much like qA did for the adenine analogues mentioned above. The second half of the thesis focuses on interbase FRET (Förster resonance energy transfer) using the new adenine analogues and the previously reported FRET-pair tCO-tCnitro. FRET is confirmed and characterized inside DNA using the three adenine donors (qAN1, qAN4 and pA) with the acceptor qAnitro. These FRET-pairs can monitor energy transfer up to 1.5 turns of DNA and are hence suitable for monitoring structural changes in short DNA. This is exemplified by a study of the effect on DNA structure by binding of netropsin, showing that the interbase FRET is sensitive to small changes in DNA structure. The previously reported tCO-tCnitro are here both incorporated into RNA and interbase FRET in RNA is measured for the first time. This is an important step since RNA, among other things, has proved to be a key player in cell regulation and hence of high interest and importance. Lastly the change in interbase FRET upon inducing a change from A- to Z-form RNA is shown to be significant, again highlighting the potential of interbase FRET in nucleic acid structure investigations.
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