Novel, monomeric cyanine dyes as reporters for DNA helicase activity.
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 Mg(2+), 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 Mg(2+). 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.
Xu
Cuiling
C
Department of Microbiology and Immunology, University at Buffalo, Buffalo, NY 14214, USA.
Losytskyy
Mykhaylo Yu
MY
Kovalska
Vladyslava B
VB
Kryvorotenko
Dmytro V
DV
Yarmoluk
Sergiy M
SM
McClelland
Sarah
S
Bianco
Piero R
PR
D001583:Benzoxazoles / Q000737:chemistry; D001667:Binding, Competitive; D002232:Carbocyanines / Q000138:chemical synthesis / Q000737:chemistry*; D004247:DNA / Q000378:metabolism; D004265:DNA Helicases / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D011806:Quinolinium Compounds / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013844:Thiazoles / Q000737:chemistry
D001583:Benzoxazoles; D002232:Carbocyanines; D004277:DNA, Single-Stranded; D005456:Fluorescent Dyes; D011806:Quinolinium Compounds; C086329:TOTO-1 thiazole orange dye; D013844:Thiazoles; C120934:cyan 2; C075296:1,1'-((4,4,7,7-tetramethyl)-4,7-diazaundecamethylene)bis-4-(3-methyl-2,3-dihydro(benzo-1,3-oxazole)-2-methylidene)quinolinium; D004247:DNA; D004265:DNA Helicases; D008274:Magnesium