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Journal of Fluorescence

, Volume 17, Issue 6, pp 671–685 | Cite as

Novel, Monomeric Cyanine Dyes as Reporters for DNA Helicase Activity

  • Cuiling Xu
  • Mykhaylo Yu Losytskyy
  • Vladyslava B. Kovalska
  • Dmytro V. Kryvorotenko
  • Sergiy M. Yarmoluk
  • Sarah McClelland
  • Piero R. BiancoEmail author
Original Paper

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.

Keywords

Cyanine dye YOYO-1 Dye-displacement DNA helicase assay RecBCD 

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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Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Cuiling Xu
    • 1
    • 2
    • 3
  • Mykhaylo Yu Losytskyy
    • 4
  • Vladyslava B. Kovalska
    • 4
  • Dmytro V. Kryvorotenko
    • 4
  • Sergiy M. Yarmoluk
    • 4
  • Sarah McClelland
    • 5
  • Piero R. Bianco
    • 1
    • 2
    • 3
    Email author
  1. 1.Department of Microbiology and ImmunologyUniversity at BuffaloBuffaloUSA
  2. 2.Department of BiochemistryUniversity at BuffaloBuffaloUSA
  3. 3.Center for Single Molecule BiophysicsUniversity at BuffaloBuffaloUSA
  4. 4.Institute of Molecular Biology and GeneticsNational Academy of Sciences of UkraineKyivUkraine
  5. 5.Marie Curie Research Institute, The Chart, OxtedSurreyUK

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