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Molecular and spectroscopic characterization of green and red cyanine fluorophores from the Alexa Fluor and AF series

Christian Gebhardt, Martin Lehmann, Maria M. Reif, Martin Zacharias, View ORCID ProfileThorben Cordes
doi: https://doi.org/10.1101/2020.11.13.381152
Christian Gebhardt
1Physical and Synthetic Biology, Faculty of Biology, Ludwig-Maximilians-Universität München, Großhadernerstr. 2-4, 82152 Planegg-Martinsried, Germany
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Martin Lehmann
2Plant Molecular Biology and Plant Metabolism, Faculty of Biology, Ludwig-Maximilians-Universität München, Großhadernerstr. 2-4, 82152 Planegg-Martinsried, Germany
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Maria M. Reif
3Theoretical Biophysics (T38), Physics Department, Technical University of Munich, München, Germany
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Martin Zacharias
3Theoretical Biophysics (T38), Physics Department, Technical University of Munich, München, Germany
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Thorben Cordes
1Physical and Synthetic Biology, Faculty of Biology, Ludwig-Maximilians-Universität München, Großhadernerstr. 2-4, 82152 Planegg-Martinsried, Germany
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  • ORCID record for Thorben Cordes
  • For correspondence: cordes@bio.lmu.de
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Abstract

The use of fluorescence techniques has had an enormous impact on various research fields including imaging, biochemical assays, DNA-sequencing and medical technologies. This has been facilitated by the availability of numerous commercial dyes, but often information about the chemical structures of dyes (and their linkers) are a well-kept secret. This can lead to problems for applications where a knowledge of the dye structure is necessary to predict (unwanted) dye-target interactions, or to establish structural models of the dye-target complex. Using a combination of spectroscopy, mass spectrometry and molecular dynamics simulations, we here investigate the molecular structures and spectroscopic properties of dyes from the Alexa Fluor (Alexa Fluor 555 and 647) and AF series (AF555, AF647, AFD647). Based on available data and published structures of the AF and Cy dyes, we present two possible structures for Alexa Fluor 555. We also resolve conflicting reports on the linker composition of Alexa Fluor 647. A comprehensive comparison between Alexa Fluor and AF dyes by continuous-wave absorption and emission spectroscopy, quantum yield determination, fluorescence lifetime and anisotropy spectroscopy of free and protein-attached dyes, supports the suggestion that the Alexa Fluor and AF dyes have a high degree of structural similarity. In addition, we compared Alexa Fluor 555 and Alexa Fluor 647 to their structural homologs AF555 and AF(D)647 in single-molecule FRET applications. Both pairs showed excellent performance in solution-based smFRET experiments using alternating laser excitation demonstrating that the AF-fluorophores are an attractive alternative to Alexa- and Cy-dyes for smFRET studies, and suggesting their usefulness for other fluorescence applications.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted November 15, 2020.
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Molecular and spectroscopic characterization of green and red cyanine fluorophores from the Alexa Fluor and AF series
Christian Gebhardt, Martin Lehmann, Maria M. Reif, Martin Zacharias, Thorben Cordes
bioRxiv 2020.11.13.381152; doi: https://doi.org/10.1101/2020.11.13.381152
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Molecular and spectroscopic characterization of green and red cyanine fluorophores from the Alexa Fluor and AF series
Christian Gebhardt, Martin Lehmann, Maria M. Reif, Martin Zacharias, Thorben Cordes
bioRxiv 2020.11.13.381152; doi: https://doi.org/10.1101/2020.11.13.381152

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