Stability, specificity and fluorescence brightness of multiply-labeled fluorescent DNA probes.
Randolph JB; Waggoner AS.
Nucleic Acids Research, 1997 Jul 15, 25(14):2923-9.
In this work, we studied the fluorescence and hybridization of
multiply-labeled DNA probes which have the hydrophilic fluorophore
1-(straightepsilon-carboxypentynyl)-1\\\'-ethyl- 3,3,3\\\',
3\\\'-tetramethylindocarbocyanine-5,5\\\'-disulfonate (Cy3) attached via either a
short or long linker at the C-5 position of deoxyuridine. We describe the
effects of labeling density, fluorophore charge and linker length upon five
properties of the probe: fluorescence intensity, the change in fluorescence
upon duplex formation, the quantum yield of fluorescence (Phif),
probe-target stability and specificity. For the hydrophilic dye Cy3, we
have demonstrated that the fluorescence intensity andPhifare maximized when
labeling every 6th base using the long linker. With a less hydrophilic dye,
a labeling density this high could not be achieved without serious
quenching of the fluorescence. The target specificity of multiply-labeled
DNA probes was just as high as compared to the unmodified control probe,
however, a less stable probe-target duplex is formed that exhibits a lower
melting temperature. A mechanism that accounts for this destabilization is
proposed which is consistent with our data. It involves dye-dye and
dye-nucleotide interactions which appear to stabilize a single-stranded
conformation of the probe.
med_UI: 97351166 ID: 41
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Hermann J. Gruber, Christoph D. Hahn, Gerald Kada, Christian K. Riener,
Gregory S. Harms, Werner Ahrer, Ý Thomas G. Dax, Ý and Hans-Gu¨ nther Knaus ý
Bioconjugate Chem. 2000, 11, 696-704
This study provides a critical examination of protein labeling with Cy3, Cy5, and other Cy dyes. Two
alternate situations were tested. (i) Antibodies were covalently labeled with Cy dye succinimidyl ester
at various fluorophore/protein ratios and the fluorescence of the labeled antibodies was compared to
that of free Cy dye. (ii) Fluorescent biotin derivatives were synthesized by derivatizing ethylenediamine
with one biotin and one Cy3 (or Cy5) residue. The fluorescence properties of these biotin-Cy dye
conjugates were examined at all ligand/(strept)avidin ratios (0 e n e 4). The results showed an
astounding discrepancy between Cy3 and Cy5: Cy3-labeled antibodies fluoresced very well, even at
high Cy3/protein ratios, and the same applied to (strept)avidin with up to four bound biotin-Cy3
conjugates. In contrast, antibodies with six covalently bound Cy5 labels (obtained with the
recommended procedure) were almost nonfluorescent, only at 2-3 Cy5 labels/IgG some moderate
fluorescence was obtained. By analogy, the biotin-Cy3 conjugate fluoresced intensely, even at high
ligand/avidin ratio, in contrast to the weakly fluorescing biotin-Cy5 conjugate. Three mechanisms
are responsible for the discrepancy between Cy3 and Cy5. (i) Attachment of Cy3 to a protein’s surface
causes an anomalous enhancement in fluorescence (by 2-3-fold) while no enhancement occurs with
Cy5. (ii) Mutual quenching of IgG-bound Cy dyes by resonance energy transfer is much more
pronounced for Cy5 labels than for Cy3. (iii) In IgG with six bound Cy5 labels, about one-third of the
labels adopt a nonfluorescent state which is characterized by a large UV-vis absorption maximum at
600 nm instead of at 650 nm. Cy3.5 was found to mimick the properties of Cy3, while Cy7, and to
some extent also Cy5.5, were similar to Cy5. In conclusion the Cy dye series is divided into two
groups: Antibodies with multiple Cy3 or Cy3.5 labels yield bright fluorescence while extensive
quenching occurs in antibodies labeled with Cy5 and Cy7.
med_UI: 20438116 ID: 44
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