Biology Reference
In-Depth Information
Modulation of the cyanines is possible through several strategies. First,
carefully chosen blocking groups can be attached to the cyanine nitrogens
to quench fluorescence.
127
An alternative strategy is to modify the poly-
methine chain with heteroatoms and attach blocking groups at those posi-
tions.
6,128
In addition, many fluorogenic cyanine constructs are based on
FRET between two different cyanine dyes (e.g., Cy3 and Cy5).
129
Finally, modulation of the fluorescence intensity by PeT is possible, but
inefficient because of the long absorption of this dye class.
130
10.2. Enzyme substrates
In addition to several FRET-based probes, unimolecular cyanine-based fluo-
rogenic probes have been developed. An example is compound
84
,whichis
an effective fluorogenic substrate for
Escherichia coli
nitroreductase. Enzyme-
catalyzed reduction of a nitro group on the dinitrophenyl ring leads to bond
cleavage and a concomitant increase in fluorescence.
127
Modulation at the poly-
methine chain is also possible. Fusing a phenol into a Cy7-like structure allows
modulation of cyanine fluorescence by attaching blocking groups onto the phe-
nolic oxygen. For example, acetate
85
exhibits low fluorescence. Cleavage of
the ester group to liberate the phenol elicits a large shift in spectral properties
(
570/715 nm) and a significant increase in quantum yield.
131
l
max
/
l
¼
em
10.3. Photoactivatable probes
Linking two cyanine dyes via a photocleavable linker can produce useful fluo-
rogenic compounds where the fluorescence is modulated by FRET. Cy3-Cy5
dimers have been conjugated to proteins on the surface of living cells.
129
In ad-
dition, the cyaninedyes exhibit photoswitchingunder certain redox conditions
that can be harnessed for super-resolution fluorescence microcopy. When
illuminated at
max
in the presence of thiol reducing agents, cyanines conju-
gates such as the actin-binding Cy5-phalloidin (
86
) can adopt a nonfluorescent
state that can be switched to a fluorescent state by illumination with shorter
wavelength light. This “dark state” could be a reduced radical
132
or a thiol
adduct of the dye.
133
Regardless, this property has been used to performstochas-
tic optical reconstruction microscopy (STORM) on a variety of samples.
134
l
10.4. Indicators
Like the cyanine-based enzyme substrates, substitution on the cyanine nitro-
gens can elicit large changes in fluorescence. Unsubstituted cyanine dyes
such as
87
(CypHer-5) are fluorescent at low pH values (p
K
a
¼
6.1) and thus
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