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Novel, Monomeric Cyanine Dyes as Reporters for DNA Helicase Activity

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Abstract

The dimeric cyanine dyes, YOYO-1 and TOTO-1, are widely used as DNA probes because of their excellent fluorescent properties. They have a higher fluorescence quantum yield than ethidium homodimer, DAPI and Hoechst dyes and bind to double-stranded DNA with high affinity. However, these dyes are limited by heterogeneous staining at high dye loading, photocleavage of DNA under extended illumination, nicking of DNA, and inhibition of the activity of DNA binding enzymes. To overcome these limitations, seven novel cyanine dyes (Cyan-2, DC-21, DM, DM-1, DMB-2OH, SH-0367, SH1015-OH) were synthesized and tested for fluorescence emission, resistance to displacement by Mg2+, and the ability to function as reporters for DNA unwinding. Results show that Cyan-2, DM-1, SH-0367 and SH1015-OH formed highly fluorescent complexes with dsDNA. Of these, only Cyan-2 and DM-1 exhibited a large fluorescence enhancement in buffers, and were resistant to displacement by Mg2+. The potential of these two dyes to function as reporter molecules was evaluated using continuous fluorescence, DNA helicase assays. The rate of DNA unwinding was not significantly affected by either of these two dyes. Therefore, Cyan-2 and DM-1 form the basis for the synthesis of novel cyanine dyes with the potential to overcome the limitations of YOYO-1 and TOTO-1.

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Abbreviations

EthD:

ethidium homodimer

DAPI:

4′,6-diamidino-2-phenylindole

YO-PRO-1:

4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(trimethylammonio)propyl]-, diiodide

YO:

oxazole yellow

TO:

thiazole orange

YOYO-1:

1,1′-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)-bis-4-[3-methyl-2,3-dihydro-(benzo-1,3-oxazole)-2-methylidene]-quinolinium tetraiodide

TOTO-1:

1,1′-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)-bis-4-[3-methyl-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene]-quinolinium tetraiodide

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Author notes

  1. Sarah McClelland

    Present address: Marie Curie Research Institute, The Chart, Oxted, Surrey, RH8 0TL, UK

Authors and Affiliations

  1. Department of Microbiology and Immunology, University at Buffalo, Buffalo, NY, 14214, USA

    Cuiling Xu & Piero R. Bianco

  2. Department of Biochemistry, University at Buffalo, Buffalo, NY, 14214, USA

    Cuiling Xu & Piero R. Bianco

  3. Center for Single Molecule Biophysics, University at Buffalo, Buffalo, NY, 14214, USA

    Cuiling Xu & Piero R. Bianco

  4. Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, 150 Zabolotnogo Street, 03143, Kyiv, Ukraine

    Mykhaylo Yu Losytskyy, Vladyslava B. Kovalska, Dmytro V. Kryvorotenko & Sergiy M. Yarmoluk

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  1. Cuiling Xu
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  2. Mykhaylo Yu Losytskyy
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Correspondence to Piero R. Bianco.

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Xu, C., Losytskyy, M.Y., Kovalska, V.B. et al. Novel, Monomeric Cyanine Dyes as Reporters for DNA Helicase Activity. J Fluoresc 17, 671–685 (2007). https://doi.org/10.1007/s10895-007-0215-z

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  • DOI: https://doi.org/10.1007/s10895-007-0215-z

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