Fluorogenic dendrons with multiple donor chromophores as bright genetically targeted and activated probes

Article abstract of DOI:10.1021/ja9099328

We have developed a class of dendron-based fluorogenic dyes (termed dyedrons) comprised of multiple cyanine (Cy3) donors coupled to a single malachite green (MG) acceptor that fluoresce only when the MG is noncovalently but specifically bound to a cognate

Full text of DOI:10.1021/ja9099328

Published on Web 07/27/2010
Fluorogenic Dendrons with Multiple Donor Chromophores as
Bright Genetically Targeted and Activated Probes
Christopher Szent-Gyorgyi,† Brigitte F. Schmidt,† James A. J. Fitzpatrick,†,§ and
Marcel P. Bruchez*,†,‡
Carnegie Mellon UniVersity, Molecular Biosensors and Imaging Center, Department of
Chemistry, 4400 Fifth AVenue, Pittsburgh, PennsylVania 15213
Received May 12, 2009; E-mail: bruchez @cmu.edu
Abstract: We have developed a class of dendron-based fluorogenic dyes (termed dyedrons) comprised of
multiple cyanine (Cy3) donors coupled to a single malachite green (MG) acceptor that fluoresce only when the
MG is noncovalently but specifically bound to a cognate single chain antibody (scFv). These cell-impermeant
dyedrons exploit efficient intramolecular energy transfer from Cy3 donors to stoichiometrically amplify the
fluorescence of MG chromophores that are activated by binding to the scFv. These chromophore enhancements,
coupled with our optimized scFv, can significantly increase fluorescence emission generated by the dyedron/
scFv complex to brightness levels several-fold greater than that for single fluorescent proteins and targeted
small molecule fluorophores. Efficient intramolecular quenching of free dyedrons enables sensitive homogeneous
(no wash) detection under typical tissue culture conditions, with undetectable nonspecific activation.
multiple small molecule fluorophores.10 These probes are often
cumbersome to prepare and, when applied to live or fixed cells
Introduction
Fluorescent detection with genetically targeted probes has
fundamentally expanded the types of questions that can be
addressed with biological microscopy and cytometry.1 The ease
of use and breadth of application of genetically expressed fluo-
rescent protein chimeras has been truly revolutionary, especially
for live-cell studies. However, the signal generated by a single
fluorescent protein or organic dye fluorophore is intrinsically limited
and typically falls well short of the requirements for robust single
molecule detection.2 Investigators often address this deficit of signal
by overexpressing receptors and other rare proteins genetically
fused to a single fluorescent protein; however, overexpression may
lead to imbalances in protein regulation, biosynthesis, or assembly.
An alternative signal amplification scheme is based on very large
genetic fusions incorporating multiple fluorescent protein copies,
but these may sterically interfere with biological function.3 Es-
sentially the same limitations apply to any of several site-specific
dye-targeting methods based on genetic fusions to peptide domains
that can be specifically modified by an in vivo enzymatic activity.4
Highly fluorescent probes may be obtained by in vitro
conjugation of antibodies and other proteins to quantum dots,5,6
phycobiliproteins,7,8 DNA multifluorophore assemblies,9 or
and tissues, generate nonspecific background fluorescence unless
excess probe is removed. Because these fully exogenous probes
are not genetically expressed by the biological system of interest,
they are difficult to deliver to extracellular and especially intra-
cellular targets in a way that allows real time monitoring and
imaging of dynamic biological functions.11
Recently we and others demonstrated the use of single-chain
variable fragment (scFv) antibodies to noncovalently constrain
chromophores of several nonrigid cyanine-family dyes, thereby
activating fluorescence hundreds- to thousands-fold.12-14 These
scFvs are examples of a broader but so far limited class of
fluorogen activating proteins (FAPs).15 FAPs that bind deriva-
tives of malachite green (MG) displayed the greatest fluorogenic
activation, up to 20 000-fold,13 primarily because of an ex-
tremely low background signal. The cell impermeant M (MG
diethyleneglycolamine, Chart 1) and cell-permeant MG-ester13
(8) Glazer, A. N.; Stryer, L. Biophys. J. 1983, 43, 383.
(9) Benvin, A. L.; Creeger, Y.; Fisher, G. W.; Ballou, B.; Waggoner, A. S.;
Armitage, B. A. J. Am. Chem. Soc. 2007, 129, 2025.
(10) Berlier, J. E.; Rothe, A.; Buller, G.; Bradford, J.; Gray, D. R.; Filanoski,
B. J.; Telford, W. G.; Yue, S.; Liu, J.; Cheung, C. Y. J. Histochem.
Cytochem. 2003, 51, 1699.
† Molecular Biosensors and Imaging Center.
(11) Michalet, X.; Pinaud, F. F.; Bentolila, L. A.; Tsay, J. M.; Doose, S.;
Li, J. J.; Sundaresan, G.; Wu, A. M.; Gambhir, S. S.; Weiss, S. Science
(Washington, D.C.) 2005, 307, 538.
‡ Department of Chemistry.
§ Current address: Waitt Advanced Biophotonics Center, Salk Institute
for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037.
(1) Giepmans, B. N. G.; Adams, S. R.; Ellisman, M. H.; Tsien, R. Y.
Science (Washington, D.C.) 2006, 312, 217.
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Armitage, B. A. J. Am. Chem. Soc. 2008, 130, 12620.
(2) McKinney, S. A.; Murphy, C. S.; Hazelwood, K. L.; Davidson, M. W.;
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K. L.; Adler, S.; Fitzpatrick, J. A. J.; Woolford, C. A.; Yan, Q.; Vasilev,
K. V.; Berget, P. B.; Bruchez, M. P.; Jarvik, J. W.; Waggoner, A.
Nat. Biotechnol. 2008, 26, 235.
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Chart 1. Structure of Dyedronsa
a Donor groups are depicted in blue, and acceptor groups in red. M, malachite green diethyleneglycolamine; CM, Cy3.29 malachite green; BCM, Bis-
Cy3.29 malachite green; TCM, Tetra-Cy3.29 malachite green. Malachite green (MG) is the chromophore shown in red.
are efficiently quenched in aqueous solution and remain so in
complex biological milieus, including in cell lysates and on and
within cells. This behavior contrasts with MG itself, which can
become moderately fluorescent in these environments.16 Fusion
proteins incorporating these FAPs can be labeled in vivo with
extremely low background fluorescence by single-step addition
of M or MG-ester, enabling new labeling applications. Employ-
ing confocal and stimulated emission depletion (STED) mi-
croscopy, we demonstrated intracellular targeting and labeling
in live or permeabilized fixed cells using a disulfide-free
cytosolic FAP-ꢀ-actin fusion.17 FAP/fluorogen complexes (fluo-
romodules) are comparable to dyes and fluorescent proteins in
brightness. Further improvements in brightness would result in
better sensitivity and lower phototoxicity under typical imaging
conditions.
Rationale
Because scFv-based fluoromodules rely on the intimate
interaction between a nonfluorescent fluorogen and a specific
(17) Fitzpatrick, J. A. J.; Yan, Q.; Sieber, J. J.; Dyba, M.; Schwarz, U.;
Szent-Gyorgyi, C.; Woolford, C. A.; Berget, P. B.; Waggoner, A. S.;
Bruchez, M. P. Bioconjugate Chem. 2009, 20, 1843.
(16) Tsien, R. Y. FEBS Lett. 2005, 579, 927.
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Table 1. Properties of L5-MG E52D Activated Dyedrons
Dyedron CM BCM
M
TCM
εmax [nm]a
642
552
140 000
0.06
8.0
1.54
2.17
<1
0.0084
551
290 000
0.05
14
2.86
3.27
4.0
0.0036
552
a
εmax [M-1 cm-1
]
79 000
0.06
4.4
530 000
0.05
ΦMGa b
,
ε × Φ/103 (brightness)
29c d
,
a
F532/F635
0.05
0.07
<1
4.95
6.89
15
e
FACS532/FACS635
KD [nM]f
g
Fquenched/Ffree
N/A
0.0037
a Determined for soluble dyedron/L5-MG E52D complex. b Quantum
yield for MG excitation peak determined with 620 nm excitation. c For
comparison, EGFP ) 32, see ref 28. d For comparison, L5-MG E52D
L91S ) 160, calculated from data in Figure 5 as ratio of fluorescence of
cell surface displayed L5-MG E52D L91S to L5-MG E52D at 30 nM
TCM. e Numerator and divisor calculated from data in Figure 3 as
(median of stained population - median of unstained control). f Deter-
mined for dyedrons binding to yeast cell surface displayed L5-MG
E52D (Figure S6). g Calculated as total absorbance-normalized fluo-
rescence (530-800 nm) of dyedron divided by total absorbance-
normalized fluorescence of Cy3.29. The MG absorbance peak was used
for dyedron normalization.
Figure 1. Binding activation of dyedrons.
binding polypeptide, additional molecules can be flexibly linked
to the fluorogen to enhance the optical properties (such as
brightness) without interfering with the fundamental binding-
mediated activation. The brightness of fluorescent probes can
be enhanced by improvements in the extinction coefficient and
improvements in the fluorescent quantum yield; the product of
these values reflects the molecular brightness. Using intramo-
lecular energy transfer from Cy3 covalently coupled to MG,
we have created compact multichromophore structures with
large extinction coefficients at Cy3 excitation wavelengths while
maintaining the MG fluorogenic properties. For dyedrons free
in solution, the absorbance of light is increased in proportion
to the number of Cy3 donor chromophores; the input energy is
efficiently transferred to the MG acceptor chromophore, where
it is nonradiatively dissipated (quenched) by unconstrained
internal rotations. For dyedrons bound to a FAP, excitation is
transferred in much the same way, except the MG acceptor
chromophore is now rigidly constrained by the peptide binding
pocket. Instead of dissipating, transferred energy is now emitted
as donor-amplified fluorescence. Previous reports have shown
that multichromophoric dendrimers have high absorbance and
multiphoton cross sections18 as well as highly efficient intramo-
lecular energy transfer,19-23 but such molecules are not con-
jugatable and do not have the inherent in vivo genetic targeting
and activation capability of this new multichromophore class.
This straightforward method for targeted activation is essential
for the biological utility of these molecules (Figure 1).
S1). For a detailed study of the dyedron properties, we employed
a 110 amino acid scFv (L5-MG E52D) derived from the original
L5-MG clone13 by directed evolution to increase affinity and
brightness when bound to M; to demonstrate that directed
evolution can generate dyedron/FAP fluoromodules with a
brightness significantly greater than that of dyes and fluorescent
proteins, we subsequently characterized additional L5-MG
mutants L91S and E52D L91S (Figure S4).
Spectral Characterization of Dyedrons Bound to L5-MG
E52D. Free in solution, all dyedrons showed >99% quenching
of Cy3 fluorescence when conjugated to MG (Table 1) and
essentially undetectable fluorescence in the spectral range
associated with MG (Figure S5), consistent with the extremely
low quantum yield of MG in the absence of an activating
polypeptide.25 The fluorescence quantum yield of the unbound
TCM dyedron was <0.0005, so direct emission of the donors is
efficiently quenched and does not interfere with detection of a
FAP-activated dyedron.
At a single concentration of dye molecule in the presence of
excess L5-MG E52D, MG-probe normalized excitation spectra
(710 nm detection) reveal that contributions of the Cy3
excitation increase in direct proportion to Cy3 number and show
that these simple modifications substantially enhance the overall
excitation cross section of the construct as compared to M alone
(Figure 2a and Table 1). The magnitude of cross-sectional
enhancement correlates well with the absorbance of Cy3 (ε )
150 000 M-1 cm-1).26 Quantum yields of all dyedrons at the
MG excitation peak were essentially constant. Corresponding
fluorescence emission spectra show almost complete transfer
(>99%) of the Cy3 excitation to far red emission from the bound
MG dye and show substantial increases in the brightness of the
probe constructs when used with increasing generations of the
dyedron (Figure 2b). Relative quantum yields at the Cy3
excitation and the MG excitation indicate that little donor
excitation is lost to competing radiative and nonradiative
Results
Compact multichromophore dyedrons in Chart 1 were
prepared by a strategy similar to that of the convergent syntheses
of Frechet24 and purified by reversed-phase liquid chromatog-
raphy, yielding branched structures with one, two, and four Cy3
donor molecules covalently and stoichiometrically decorating
the periphery of the molecule and a single MG quenching group
at the base of the dyedron (Scheme S1, Supporting Information
‘Dyedron synthesis and characterization’, Figures S1-S3, Table
(18) Mongin, O.; Krishna, T. R.; Werts, M. H. V.; Caminade, A. M.;
Majoral, J. P.; Blanchard-Desce, M. Chem. Commun. 2006, 2006, 915.
(19) Balzani, V.; Ceroni, P.; Gestermann, S.; Gorka, M.; Kauffmann, C.;
Maestri, M.; Vo¨gtle, F. ChemPhysChem 2000, 1, 224.
(20) Devadoss, C.; Bharathi, P.; Moore, J. S. J. Am. Chem. Soc. 1996,
118, 9635.
(25) Babendure, J. R.; Adams, S. R.; Tsien, R. Y. J. Am. Chem. Soc. 2003,
125, 14716.
(21) Serin, J. M.; Brousmiche, D. W.; Frechet, J. M. J. J. Am. Chem. Soc.
2002, 124, 11848.
(26) Mujumdar, R. B.; Ernst, L. A.; Mujumdar, S. R.; Lewis, C. J.;
Waggoner, A. S. Bioconjugate Chem. 1993, 4, 105.
(27) Hung, S.-C.; Ju, J.; Mathies, R. A.; Glazer, A. N. Anal. Biochem. 1996,
243, 15.
(22) Serin, J. M.; Brousmiche, D. W.; Fre´chet, J. M. J. Chem. Commun.
2002, 2002, 2605.
(23) Balzani, V.; Ceroni, P.; Maestri, M.; Vicinelli, V. Curr. Opin. Chem.
Biol. 2003, 7, 657.
(28) Patterson, G. H.; Knobel, S. M.; Sharif, W. D.; Kain, S. R.; Piston,
D. W. Biophys. J. 1997, 73, 2782.
(24) Hawker, C. J.; Frechet, J. M. J. J. Am. Chem. Soc. 1990, 112, 7638.
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Figure 2. Fluorescence spectroscopy of dyedron/L5-MG E52D complex.
(a) Relative fluorescence excitation of dyedron/L5-MG E52D complexes
(710 nm emission). (b) Relative emission of dyedron/L5-MG E52D
complexes (514 nm excitation). Spectra have been normalized to the MG
excitation peak.
Figure 3. Flow cytometric analysis of dyedron-labeled yeast by exciting
donor or acceptor. Saccharomyces cereVisiae cells expressing L5-MG E52D
on their surface were analyzed as described.13 Two aliquots of each stained
population and an unstained control (U) were respectively analyzed, with
donor (532 nm) or acceptor (635 nm) excitation, and collecting emission
through a 675/50 nm bandpass filter. Position of unstained cells is shown
by thin lines. Each analysis comprised 100 000 cells.
processes (Table S2). These observations support the concept
that even inherently nonfluorescent or self-quenched donors in
dyedrons could produce highly efficient sensitizing structures
for bright fluorescence.10,27 Regardless of mechanism, dyedrons
are efficiently quenched when unbound and efficiently activated
for both direct and donor amplified fluorogen emission when
bound.
When bound to the E52D FAP, the absorbance of M and all
dyedron MG acceptors increase nearly 2-fold and their absor-
bance maxima red-shift and coalesce at ∼642 nm, suggesting
that acceptor photophysical properties are specifically modulated
by the FAP binding pocket and are largely independent of the
donors. In contrast, absorbance spectra at donor wavelengths
of FAP-bound dyedrons and free Cy3 have similar features,
suggesting that Cy3 photophysics are not greatly altered (Figure
S7 and Figure S7 discussion), although some evidence of
donor-donor interaction is evident in the BCM and TCM
complexes.
Dyedron/L5-MG E52D Fluoromodules Expressed on Live
Cell Surfaces. In vitro spectroscopic properties of L5-MG E52D
fluoromodules are recapitulated when dyedrons (300 nM) are
directly added to suspensions of live yeast cells expressing the
fluorogen activating scFv as a fusion protein on the cell wall.13
Flow cytometry reveals step increases in brightness when excited
at 532 nm and nearly constant brightness when excited at 635
nm (Figure 3), corresponding well to the differences seen in
the excitation spectra and the consistent quantum yields
measured at 620 nm excitation. Stained samples contain a
subpopulation of nonfluorescent cells due to loss of an scFv-
encoding plasmid.13 Controls show virtually no fluorescence
generated by dyedrons on cell surfaces in the absence of
expressed FAPs (Table S3). Analysis of the staining ratio
between yeast cells excited at 635 nm vs 532 nm reveals an
increase in specific brightness by a factor of ∼2 (n ) 1), 3
(n ) 2), and 7 (n ) 4) in the (Cy3)nMG construct (Tables 1
and S3). Hence, this approach increases molecular brightness
in vitro and in vivo in direct correlation with the enhanced
extinction provided by the donor array.
Dyedron-mediated signal amplification can also be applied
to live cell fluorescence microscopy. Yeast cells expressing the
L5-MG E52D fusion protein13 on their surface were imaged
under a laser scanning confocal microscope (Figure 4). Yeast
cells are specifically labeled on their surface. Virtually no
fluorescence is detected from cells not expressing FAPs or from
intercellular regions. The relative signal levels of M, CM, and
TCM treated cells imaged at 561 and 633 nm excitation follow
the same trends observed in solution fluorimetry and flow
cytometry (Figures 2, 3).
Further Improving Dyedron Fluoromodules by Directed
Evolution. The M/L5-MG E52D fluoromodule has a modest
quantum yield, yet the TCM dyedron can amplify its signal to
give a calculated molecular brightness similar to that of EGFP
and most small molecule protein tags (Table 1). The L5-MG
E52D FAP was created by applying directed evolution methods
to the very low quantum yield progenitor L5-MG FAP.13 An
error prone polymerase chain reaction using L5-MG DNA as a
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A R T I C L E S
Figure 5. Improvement of TCM fluoromodule affinity and brightness by
directed evolution. Live yeast cells expressing surface-displayed L5-MG
FAPs carrying indicated point mutations were assayed on a fluorescence
microplate reader. Values for binding affinity (Kd in nM) and relative
brightness (Bmax in arbitrary fluorescence units) are shown. The E52D and
L91S mutations independently increase fluoromodule brightness, but only
E52D confers tighter binding. The double mutant displays enhanced
brightness and tighter binding.
Figure 4. Fluorescence imaging of yeast cells surface displaying L5-MG
E52D. Live yeast cells were imaged in PBS+ in the presence of 100 nM
TCM, CM, or M on a Zeiss 510 MetaNLO confocal microscope using
differential interference contrast (DIC) or fluorescence with donor (561 nm)
or acceptor (633 nm) excitation and identical 650-710 nm BP emission
settings (see Table S4 for other imaging parameters).
so that only dyedrons bound to their biological target contribute
significantly to the overall fluorescence. The molecular weight
of these synthetic macromolecules remains small; dyedrons
constitute a minor portion of fluoromodule mass, which is
comparable to the size of GFP (∼27 kDa), and are significantly
smaller than fully exogenous conjugates currently in use as very
bright fluorescent probes. Dyedrons greatly enhance the fluo-
rescence of fluorogen/FAP complexes, as shown by the flow
cytometry, microplate assays, and microscopy described here.
The TCM/L5-MG E52D L91S fluoromodule has an estimated
brightness value (160) that is nearly 5-fold higher than those
of EGFP (Table 1) and the red fluorescent biarsenical complex
(ReAsH ) 34)29 and ∼10-fold brighter than the best available
monomeric red fluorescent protein (mCherry ) 16).30 This
improved dyedron fluoromodule has a long Stokes-shift and far-
red emission (664 nm versus 610 nm for mCherry and ReAsH),
ensuring that this probe provides substantial improvements in
sensitivity.12,31 With dyedrons, binding of the fluorogen to the
target peptide also brings the donor array into the binding site
vicinity, reducing the overall peptide-fluorogen affinity (Table
1), but the stability of these complexes remains high (<20 nM
for the E52D mutant). Such functional variation can be corrected
or exploited by the directed evolution of scFvs or other rec-
ognition scaffolds.
template was used to generate a population of plasmid encoded
FAPs carrying random mutations, and FAP mutants displaying
increased brightness and binding affinity when bound to M were
selected from among this population by FACS screening of cell
surface expressed FAPs.13
In addition to the L5-MG E52D FAP studied here, we
characterized L5-MG L91S, which contains a single point
mutation that increases the quantum yield of the M/L5-MG
fluoromodule several-fold but does not increase the binding
affinity to M (data not shown). We found that the L91S and
E52D mutations in combination behave additively to create a
FAP that binds TCM with similar affinity to L5-MG E52D and
yields ∼5-fold greater fluorescence when assayed on a micro-
plate reader using live yeast (Figures 5 and S4). Yeast cells
displaying L5-MG or evolved FAPs carrying the depicted point
mutations were assayed in a 96-well microplate (554 nm
excitation/660 nm emission) over a 1000-fold concentration
range of TCM. Data were normalized to the number of yeast-
displayed FAPs, independently determined by flow cytometric
analysis of cells labeled with fluorescent antibody bound to the
c-myc epitope present on each scFv (data not shown).13
The improved fluorescence properties of L5-MG E52D L91S
are evident when imaging the surface of live yeast in PBS buffer
using much lower concentrations of TCM (Figure 6). Yeast cells
surface-displaying this FAP can also be readily imaged with
TCM in yeast growth media (Figure S8), facilitating real time
experiments on actively metabolizing cells.
Variation in donor chemistry can thus be combined with
variation in fluorogen/peptide interaction to improve dyedron
properties. One can select for improved fluorogen binding
affinity and quantum yield in the context of a given donor array.
The donor array can be designed to enhance the extinction
coefficient but may also be designed to improve other optical
properties, such as enhancing the multiphoton cross section of
specific fluorogens, or providing targeted activation of photo-
chemically or environmentally sensitive dyes.
Discussion
These dyedrons represent a new class of fluorescent detection
reagent, where a genetically targetable acceptor chromophore
is enhanced for efficient excitation by energy transfer from
covalently attached donor molecules. Importantly, intrinsic donor
chromophore emission is efficiently quenched in free solution,
(29) Martin, B. R.; Giepmans, B. N. G.; Adams, S. R.; Tsien, R. Y. Nat.
Biotechnol. 2005, 23, 1308.
(30) Shaner, N. C.; Campbell, R. E.; Steinbach, P. A.; Giepmans, B. N. G.;
Palmer, A. E.; Tsien, R. Y. Nat. Biotechnol. 2004, 22, 1567.
(31) Chao, G.; Lau, W. L.; Hackel, B. J.; Sazinsky, S. L.; Lippow, S. M.;
Wittrup, K. D. Nat. Protoc. 2006, 1, 755.
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without washes or other treatments, distinguishing these
fluorogens from other small molecule labeling methods that
have been applied to membrane proteins.4,13,34-45 Enhanced
sensitivity of expressible probes will reduce the need for high
level overexpression in cell biological investigations33 and
may thus reduce artifacts related to expression level. Injection
into transgenic animals is potentially feasible because these
dyedrons are small enough to efficiently penetrate intercel-
lular space in tissues.46
The dyedrons described here currently provide optimal signal
enhancement for applications such as flow cytometry, microplate
assays, and imaging formats where individual fluoromodules
are subjected to moderate excitation flux. Future work will
address characterizing and improving dyedron fluoromodule
photostability by addition of electron-withdrawing groups to Cy3
donors,14 by the use of other donor chromophores, and by
directed evolution of the FAP to increase the on/off rate of
dyedron binding, thereby continuously regenerating functional
fluoromodules.13,14
Genetically expressed fluorescent protein fusions are widely
used to study biological functions in the cytoplasm and other
intracellular reducing environments. Current fluorogen-activating
scFvs contain internal disulfide linkages that compromise
function in these intracellular compartments, but directed
evolution may be employed to remove such disulfides and adapt
scFvs to intracellular function.17,47 It would then be desirable
to introduce dyedrons into the cell for maximum utility. Several
approaches to transmembrane delivery are available, including
microinjection, pore formation, modifying dyedron physico-
chemical properties, addition of transport signals to dyedrons,
or creation of dyedron-carrier vesicles or emulsions able to fuse
with the plasma membrane. Microinjection of the TCM dyedron
into live mammalian cells showed no detectable fluorescence
up to 50× over the Kd (Figure S9). The low nonspecific
activation of dyedrons indicates that the main barrier to
intracellular use may be a problem of delivery, not one of
specificity.
Figure 6. Live cell surface imaging with improved fluoromodules. Yeast
cells expressing L5-MG carrying E52D (a) or E52D L91S (b) mutations
were imaged in PBS+ on a Zeiss 510 MetaNLO confocal microscope
with identical donor (561 nm) excitation/650-710 nm BP emission
settings using the indicated TCM concentrations. Scan profiles show
that fluorescence of the E52D L91S double mutant at 10 nM TCM is
∼5-fold greater than E52D fluorescence at 10-fold higher TCM
concentration. The brighter signal seen at lower TCM concentration with
the double mutant is consistent with its improved brightness and binding
affinity.
(34) Keppler, A.; Gendreizig, S.; Gronemeyer, T.; Pick, H.; Vogel, H.;
Johnsson, K. Nat. Biotechnol. 2002, 21, 86.
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The membrane impermeant nature of these dyedrons makes
them ideal for studying a wide range of biological functions
involving plasma membrane proteins that have exposed
extracellular domains available for genetic fusion. Among
these are receptors that mediate intracellular signaling such
as G-protein coupled receptors32 and receptor tyrosine
kinases, ion and metabolite transport channels, and cellular
recognition and adhesion proteins. Exclusive labeling of
extracellular domains confines detection of these proteins to
the site of their biological function. In contrast, fusions of
fluorescent proteins to these same extracellular domains
would also be subject to detection during biosynthesis and
intracellular transport, generating a background signal un-
related to function at the cell surface.13,33 M and its dyedron
counterparts can be added to cell culture media directly
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11108 J. AM. CHEM. SOC. VOL. 132, NO. 32, 2010

Genetically Activated Fluorogenic Dendrons
A R T I C L E S
Acknowledgment. NIH TCNP 5U54RR022241 and R01-NIH
1R01GM086237 has supported C.S.G., B.F.S., J.A.J.F., and M.P.B.
in this work. We thank Susan Andreko for preparation of protein,
Carol Woolford and Sarah Capcek for scFv mutagenesis, Mark Bier
for Mass Spectroscopy, Roberto Gil for help with NMR, Yehuda
Creeger for assistance with flow cytometry, and Bruce Armitage
for a helpful reading of the manuscript.
Supporting Information Available: Scheme S1, Dyedron
synthesis and characterization, Figures S1-S9, Tables S1-S4,
Spectroscopy and microscopy methods, References. This ma-
terial is available free of charge via the Internet at http://
pubs.acs.org.
JA9099328
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