Blue fluorescent dye-protein complexes based on fluorogenic cyanine dyes and single chain antibody fragments

Article abstract of DOI:10.1039/c0ob00444h

Fluoromodules are complexes formed upon the noncovalent binding of a fluorogenic dye to its cognate biomolecular partner, which significantly enhances the fluorescence quantum yield of the dye. Previously, several single-chain, variable fragment (scFv) antibodies were selected from a yeast cell surface-displayed library that activated fluorescence from a family of unsymmetrical cyanine dyes covering much of the visible and near-IR spectrum. The current work expands our repertoire of genetically encodable scFv-dye pairs by selecting and characterizing a group of scFvs that activate fluorogenic violet-absorbing, blue-fluorescing cyanine dyes, based on oxazole and thiazole heterocycles. The dye binds to both yeast cell surface-displayed and soluble scFvs with low nanomolar K_d values. These dye-protein fluoromodules exhibit high quantum yields, approaching unity for the brightest system. The promiscuity of these scFvs with other fluorogenic cyanine dyes was also examined. Fluorescence microscopy demonstrates that the yeast cell surface-displayed scFvs can be used for multicolor imaging. The prevalence of 405 nm lasers on confocal imaging and flow cytometry systems make these new reagents potentially valuable for cell biological studies.

Full text of DOI:10.1039/c0ob00444h

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Organic &
Biomolecular
Chemistry
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PAPER
Blue fluorescent dye-protein complexes based on fluorogenic cyanine dyes and
single chain antibody fragments†
Kimberly J. Zanotti,a Gloria L. Silva,a Yehuda Creeger,c Kelly L. Robertson,a Alan S. Waggoner,b,c
Peter B. Bergetb,c and Bruce A. Armitage*a,c
Received 18th July 2010, Accepted 18th November 2010
DOI: 10.1039/c0ob00444h
Fluoromodules are complexes formed upon the noncovalent binding of a fluorogenic dye to its cognate
biomolecular partner, which significantly enhances the fluorescence quantum yield of the dye.
Previously, several single-chain, variable fragment (scFv) antibodies were selected from a yeast cell
surface-displayed library that activated fluorescence from a family of unsymmetrical cyanine dyes
covering much of the visible and near-IR spectrum. The current work expands our repertoire of
genetically encodable scFv-dye pairs by selecting and characterizing a group of scFvs that activate
fluorogenic violet-absorbing, blue-fluorescing cyanine dyes, based on oxazole and thiazole heterocycles.
The dye binds to both yeast cell surface-displayed and soluble scFvs with low nanomolar Kd values.
These dye-protein fluoromodules exhibit high quantum yields, approaching unity for the brightest
system. The promiscuity of these scFvs with other fluorogenic cyanine dyes was also examined.
Fluorescence microscopy demonstrates that the yeast cell surface-displayed scFvs can be used for
multicolor imaging. The prevalence of 405 nm lasers on confocal imaging and flow cytometry systems
make these new reagents potentially valuable for cell biological studies.
resulting in significant fluorescent enhancement.9,10 The FAP scFvs
Introduction
can be genetically encoded and fused to a protein of interest to
The development of genetically encodable fluorescent protein tags
be used in a manner similar to GFP. When a fluorogenic dye
has transformed the field of cellular imaging.1 Green fluorescent
accesses the site where the FAP is expressed, the dye is bound by
protein (GFP)2,3 is a prominent example, and can be attached
the FAP and fluorescence is observed. Unlike GFP, the timing of
the fluorescence signal can be controlled based on when the dye
is added to the FAP. The fluorescent signal of the fluoromodule
as a fusion tag and used to track a protein of interest in
vivo.4–7 Although GFP and its derivatives have become standard
cellular imaging tools, it can be problematic to precisely tune
can also be restored after photobleaching by simply adding fresh
dye to exchange out the bleached dye,11 something which is not
possible when the chromophore is covalently integrated within the
protein.7
properties such as fluorescence color and brightness because the
chromophore is covalently embedded within the protein. Our
Center has recently created a class of novel fluorophores using
single-chain variable fragment (scFv) antibodies and fluorogenic
dyes.8 These dye-protein pairs are called fluoromodules, in which
the fluorescence-activating protein (FAP) binds noncovalently to
a target fluorogenic dye. In a conformationally unconstrained
environment, such as fluid solution, the dye loses energy through
torsional movement around a conjugated methine bridge and is
nonfluorescent. However, in a constrained environment such as an
scFv’s binding pocket, torsional movement of the dye is restricted,
Our fluoromodule systems are readily fine-tuned by rationally
designing new fluorogenic dyes with the desired wavelength and
photostability properties. If new dyes do not bind to existing FAPs
obtained from prior selections, new FAPs that activate these dyes
are selected using a yeast surface-displayed scFv library.12 One of
our Center’s goals is to develop fluoromodules covering the entire
visible and near-IR spectrum, allowing for multicolor labeling
at any desired absorption/emission wavelengths. Previous work
resulted in the selection and characterization of FAPs that bind
to and activate the dyes thiazole orange, malachite green, and
dimethylindole red (DIR).8,11,13–15 Although most scFvs exhibited
high specificity and selectivity for only their target dye, one DIR-
binding scFv was capable of activating a family of unsymmetrical
cyanine dyes spanning most of the visible and near-IR spectrum
with low nanomolar dissociation constants (Kd).13 However, the
promiscuous scFv bound much weaker to a violet-absorbing dye.
Our goal in the current study was to create a new family of
aDepartment of Chemistry, Carnegie Mellon University, 4400 Fifth Avenue,
Pittsburgh, PA, 15213, USA. E-mail: army@cmu.edu; Fax: +1 412-268-
1061; Tel: +1 412-268-4196
bDepartment of Biological Sciences, Carnegie Mellon University, 4400 Fifth
Avenue, Pittsburgh, PA, 15213, USA
cDepartment of Molecular Biosensor and Imaging Center, Carnegie Mellon
University, 4400 Fifth Avenue, Pittsburgh, PA, 15213, USA
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c0ob00444h
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Chart 1 Fluorogenic dye structures.
blue fluoromodules with low nanomolar Kds and high quantum
yields (ff).
leading to significant background fluorescence as shown in the
ESI.† Instead of using PO-PRO-1 as our target dye, we synthesized
a variant of the blue dye Cyan47.18 We chose to re-christen Cyan47
with the more descriptive name of oxazole thiazole blue (OTB).
The new dye used for our fluoromodules, OTB-SO3, differs from
the original OTB by the addition of a sulfonate group, which was
introduced to reduce the nonspecific binding to DNA reported
previously for OTB.18 A similar strategy was used by our group to
inhibit DNA binding by DIR.19
Results
Rationale
The structures of the fluorogenic dyes used in these studies are
given in Chart 1. Previously, our group reported a promiscuous
scFv capable of activating a variety of structurally similar unsym-
metrical cyanine dyes spanning much of the visible spectrum.13
Dyes containing various heterocycles and conjugated methine
bridge lengths were activated by the promiscuous FAP with
low nanomolar Kds. However, the blue dye studied, PO-PRO-
1, had a significantly higher dissociation constant of 712 nM
with the yeast cell surface-displayed FAP. PO-PRO-1 was also
structurally dissimilar from the other dyes in that it had a
pyridinium heterocycle in place of the normal quinolinium; this
may have altered pi-stacking, van der Waals attractions, and/or
hydrophobic interactions that help to stabilize the complexes
formed with the higher affinity dyes.
Synthesis and characterization of OTB-SO3
The sulfonated version of OTB was synthesized as shown in
Scheme 1. Briefly, 2-methylbenzoxazole and 2-(methylthio)-1,3-
benzothiazole were alkylated with methyltosylate and propane-
sultone, respectively. The two half-dyes were then condensed in
the presence of triethylamine to give the dye. (Full experimental
details are provided in the ESI†)
The absorption and fluorescence emission spectra of OTB-SO3
are given in Fig. 1. The absorption spectrum features a maximum
at 400 nm (e = 92,400 M-1cm-1) and a vibronic shoulder at
385 nm. The dye’s absorption properties are unchanged upon
the addition of 100 mM calf thymus DNA to buffer. Meanwhile,
OTB-SO3 exhibits low fluorescence in buffer and only a 2.3-fold
enhancement in the presence of DNA. However, fluorescence is
Because the Kd of PO-PRO-1 and the promiscuous scFv was
significantly higher than desired, we decided to create an entirely
new blue fluoromodule with higher affinity. PO-PRO-1 is not
ideal for intracellular use because it can intercalate into DNA,16,17
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Scheme 1
Fig. 1 A. UV-vis spectra of OTB-SO3 in 10 mM sodium phosphate buffer, 100 mM NaCl (pH 7) and in the presence of 100 mM calf thymus DNA. B.
Fluorescence spectra of OTB-SO3 in 10 mM sodium phosphate buffer (pH 7) with 100 mM NaCl, in the presence of 100 mM base pairs calf thymus DNA
or in 90% glycerol. Samples were excited at 380 nm.
enhanced 56-fold in 90% glycerol solution, demonstrating the
fluorogenic potential of this unsymmetrical cyanine dye.
Both PO-PRO-1 and OTB are also fluorogenic, but their
fluorescence is activated by DNA as well as glycerol (ESI†).
The much lower fluorescence activation of OTB-SO3 by DNA
is presumably due to the dye’s negatively charged sulfonate group
repelling the phosphate backbone. By preventing interactions with
endogenous DNA, the background fluorescence of the dye should
be significantly reduced in an intracellular environment, which is
an important property for future applications of fluoromodules
based on this dye.
8 ¥ 108 recombinant human scFvs and was developed by Dane
Wittrup of MIT.12,20 These non-immune scFvs were made from
cDNA and are representative of a na¨ıve germline repertoire. The
scFvs are encoded in the pPNL6 plasmid and expressed as fusion
proteins to the yeast cell surface protein Aga2p. Each two-domain
scFv is ca. 250 amino acids long and consists of a heavy and light
chain joined together by an artificial (SerGly4)3 linker.
The binding of OTB-SO3 to one or more of these yeast surface-
displayed scFvs should result in enhanced blue fluorescence, which
can be monitored via flow cytometry. The library was mixed with
OTB-SO3 and sorted using Fluorescence-Activated Cell Sorting
(FACS) to select those cells having the brightest blue fluorescence
and FAP expression levels (ca. top 2% of the population). For the
initial round of flow cytometry, ca. 2.4 ¥ 108 cells, which covers
approximately a quarter of the original library’s diversity, were
sorted after a half hour incubation with 1 mM OTB-SO3. The
FAP Selection
The na¨ıve yeast surface-display library used to select the protein
components of our OTB-SO3 fluoromodules consisted of ca.
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selected cells were taken through four additional rounds of FACS
sorting in the presence of OTB-SO3. Individual cells were then
sorted onto an agar induction plate.21 Data from each round of
sorting are provided in the ESI.† The twelve brightest clones were
selected for further analysis. Sequencing results showed that 7
of these clones were unique. Preliminary analysis showed that
one of the clones had a comparatively large Kd (>1 mM), so this
clone was discarded. The other 6 clones were analyzed in detail;
their light and heavy chain sequences are provided in the ESI.†
Little homology was present among the FAPs’ complementarity
determining regions.
Fig. 2 depicts flow cytometry histograms of blue fluorescence
signal of the unsorted na¨ıve yeast library and clone A5. In the
unsorted library, very little blue signal is seen. In contrast, for clone
A5, the average blue fluorescence is greatly enhanced, indicating
that yeast expressing this protein on their surface significantly
activate OTB-SO3. A similar trend was seen for the other five yeast
surface-displayed OTB-SO3-binding clones (data not shown).
Fluoromodule characterization
The clones were put into a protein expression vector and soluble
hexahistidine-tagged versions of the FAPs were purified on a
Ni2+ column following standard protocols.12 Absorption and
fluorescence spectra for OTB-SO3 with soluble scFvs are shown
in Fig. 3. The binding of the clones to OTB-SO3 causes up to an
11 nm red-shift in the maximum absorbance wavelength relative to
dye in buffer. A similar trend is seen in the fluorescence emission
spectra of the OTB-FAP complexes, with the emission maxima
ranging from 422 nm with clone A6 to 440 nm with clone H10.
The emission profile for OTB-SO3 with H10 differs from the
other fluoromodules. In the case of H10, a shoulder is observed
on the short wavelength side of the main emission band, whereas
the shoulder appears on the long wavelength side of the peak for
the other fluoromodules as well as for OTB-SO3 in glycerol. The
variation in the relative intensities of these two bands could reflect
differences in the rigidity and/or conformation of the dye when
bound to the proteins, suggesting that the spectral profiles might
be temperature dependent. However, the fluorescence spectrum
of the OTB-SO3/H10 complex exhibited minimal change in
the range of 10–45 ◦C, where the protein begins to denature
(ESI†). In separate experiments, excitation spectra recorded while
monitoring emission at 425 nm and 450 nm yielded identical
profiles, indicating that a single emitting species is present (data
not shown).
We also examined the pH dependence of the OTB-SO3/H10
fluorescence (ESI†). The fluorescence intensity exhibits consider-
able variation, with 3–4-fold enhancements (relative to free dye)
at acidic pH, compared with 15–20-fold enhancements at neutral
and alkaline pH.
Dissociation constants were determined for OTB-SO3 by flu-
orescence titration in the presence of either the yeast surface-
displayed or soluble FAPs. Dye was titrated into either 107 cells
or 50 nM soluble protein; representative binding curves for clone
A5 are shown in Fig. 4 with results for other clones provided
in the ESI.† Control experiments were also run where dye was
titrated into either buffer or into yeast cells where expression of the
scFv-containing plasmid was not induced. Minimal fluorescence
Fig. 2 Flow cytometry histograms of blue fluorescence emission of the
unsorted na¨ıve yeast library (gray) and clone A5 (blue) in the presence of
1 mM OTB-SO3. Samples were excited with a 405 nm laser.
Fig. 3 (A) Absorption spectra of 1.6 mM OTB-SO3 and 3.2 mM soluble scFv. The absorption spectrum for B11 is not shown to reduce the complexity
of the figure; the profile is very similar to that of the dye in buffer. (B) Fluorescence spectra of 2.25 mM OTB-SO3 + 4.5 mM soluble scFv. Spectra were
corrected for differences in absorbance at the excitation wavelength (370 nm).
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Table 1 Absorbance and fluorescence emission maxima, equilibrium dissociation constants (Kd) with scFvs either expressed on the yeast cell surface
or in solution, fluorescence enhancement, and fluorescence quantum yields (Uf ) for the dyes bound to soluble scFv. (Fluorescence enhancement values
were calculated for soluble protein relative to the dye in buffer and were not corrected for differences in absorbance.)
scFv + OTB-SO3
Abs lmax/nm
Em lmax/nm
Yeast surface Kd/nM
Protein Kd/nM
Fluor. Enhance.
Uf
A5
411
400
400
410
405
410
431
422
424
426
440
435
109.5 5.5
6.9 1.4
13.0 0.8
12.3 0.7
119.0 6.5
166.9 4.2
93.0 2.9
9.4 0.5
7.7 0.3
12.9 0.8
72.4 4.1
63.3 2.0
52.6
9.73
20.2
49.2
54.5
50.7
0.841
0.047
0.353
0.904
1.00
A6
B11
D10
H10
J10
0.877
scFv + t-butyl-OTB-SO3
A5
413
413
625
434
437
650
42.3 1.4
39.4 1.5
24.4 1.6
61.5 3.1
31.2 1.8
21.2 1.0
36.6
40.0
8.2
0.827
0.605
0.257
scFv + t-butyl-OTB-CO2
A5
scFv + DIR
J10
Fig. 4 Fluorescence titration of OTB-SO3 into (A) yeast surface-displayed A5 or (B) soluble A5. Samples were fit to a one-site binding equation. For A,
dye was titrated into 107 cells expressing scFvs (squares), cells not expressing scFvs (circles), or buffer (triangles). Samples were excited at 401 nm. For B,
dye was titrated into 50 nM protein (squares) or buffer (triangles), and samples were excited at 380 nm and normalized to the fluorescence maximum.
signal was seen for both controls, demonstrating that little intrinsic
background fluorescence or nonspecific binding to the yeast cell
surface is present. All Kd values were relatively low at <200 nM
(Table 1). The Kds for OTB-SO3 with the soluble proteins were
slightly lower than for the cell surface-displayed versions. This
may be due to partial blockage of the dye binding site by the cell
surface environment, misfolding of the protein on the cell surface,
or a small amount of nonspecific binding of the dye to the cell
surface.
Quantum yield values were also determined for OTB-SO3 with
an excess of soluble protein at concentrations much higher than
their Kd values (Table 1). A wide range of quantum yields was
observed, with four of the OTB-SO3 fluoromodules having ff >
0.8, which is significantly higher than was observed for our
previously reported fluoromodules.8,11,13 All FAP-dye pairs had
quantum yields substantially higher than those of the dye in
buffer or in the presence of 100 mM calf thymus DNA (data
not shown). Clone H10 was especially notable for its very high
quantum yield of ~1.00, which was verified multiple times and
measured against two different standards (quinine sulfate and
9,10-diphenylanthracene). Interestingly, there is little correlation
between affinity and quantum yield. For example, FAPs A6 and
D10 bind to OTB-SO3 with similar Kd values (9.4 and 12.9 nM,
respectively) but their ff values differ by nearly 20-fold (0.047 vs.
0.904).
Promiscuity
Our previous work with the DIR-binding FAP K7 demonstrated
a high degree of promiscuity in that the protein was able to
bind a wide range of cyanine dyes, giving rise to a “rainbow”
of fluoromodules.11,13 We performed similar studies with the
OTB-SO3-binding scFvs selected in this study. This included
experiments with the other OTB dyes shown in Chart 1: t-
butyl-OTB-SO3 and t-butyl-OTB-CO2 both have a t-butyl group
attached to position 5 on the benzoxazole heterocycle. (Synthesis
and characterization of these dyes are described in the ESI.†) This
substituent was added to the ring in order to suppress intercalation
of the dye into DNA. However, fluorescence spectra shown in
Fig. 5 indicate that these dyes actually exhibit slightly greater DNA
binding based on the larger fluorescence enhancement compared
with OTB-SO3 (Fig. 1).
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Fig. 5 Fluorescence enhancement of samples excited at 380 nm relative to dye in buffer of (A) t-butyl-OTB-SO3 and (B) t-butyl-OTB-CO2 in 10 mM
sodium phosphate buffer, 100 mM NaCl (pH 7), in the presence of 100 mM calf thymus DNA, in 90% glycerol, and with A5 protein. Spectra were
corrected for differences in absorbance at the excitation wavelength.
The addition of the t-butyl group does not significantly affect the
absorbance and fluorescence maxima relative to OTB-SO3. Only
one of the six scFvs, A5, was able to fluorogenically activate all
three OTB variants. The other proteins showed no ability to bind
to the t-butyl-modified dyes. A5 bound with 1.5 and 3-fold lower
Kd values to t-butyl-OTB-SO3 and t-butyl-OTB-CO2 respectively,
compared to OTB-SO3. This suggests that A5, unlike the other
scFvs, has extra room in its binding pocket that allows the protein
to accommodate the bulky t-butyl group. It is also possible that
the OTB dyes are bound to A5 in a different orientation compared
with the other FAPs. High-resolution structural information
would provide more information on the shape complementarity
of these protein-dye complexes. High ff values were observed for
all three modified OTB dyes bound to soluble A5 (Table 1).
The ability of these scFvs to activate the parent unmodified OTB
was also studied. All 6 soluble FAPs were able to bind to OTB with
a 10-fold to 45-fold fluorescence enhancement relative to dye in
buffer (ESI†). This indicates that the alkyl sulfonate substituent on
OTB-SO3 is not essential for binding. However, unmodified OTB
readily interacts with double-stranded DNA,18 which would result
in a high background if used as an intracellular label or sensor.
Because of this, the modified OTB dyes are better candidates for
further fluoromodule development.
The promiscuity of A5 with the t-butyl-OTB dyes led us
to test whether any of the OTB-binding proteins were capable
of activating DIR. Unlike the OTB dyes, DIR has a bulky
dimethylindole heterocycle and a 3-carbon methine bridge. In
spite of these substantial changes in dye structure, FAP J10 still
fluorogenically activates DIR (Fig. 6; absorption spectra are given
in the ESI†). Surprisingly, soluble J10 bound DIR 3-fold better
than OTB-SO3. The ff of DIR bound to soluble J10 was found to
be 0.257, which is comparable to the ff previously determined for
an scFv intentionally selected to bind DIR (ff = 0.33).13
Fig. 6 Fluorescent enhancement of samples excited at 570 nm containing
750 nM DIR in the presence of 1.5 mM J10 FAP relative to fluorescence
of the dye in buffer. Spectra were corrected for differences in absorbance
at the excitation wavelength.
these samples are provided in the ESI.† The same cells are
labeled with both dyes, indicating that this single FAP can activate
fluorogenic dyes at both ends of the visible spectrum. The sequence
of J10 was unique compared to all of the FAPs previously selected
for DIR,13 even though the same na¨ıve library was used at the
start of prior DIR selections. This further illustrates the diversity
of the unsorted original library and suggests that repeating the
selection process could yield additional new FAPs that activate a
given target dye.
In contrast to the promiscuous binding of J10, we used a
previously developed dye-FAP pair (H6-MG, which activates the
red-emitting dye malachite green)8 and the newly selected H10
scFv to demonstrate the possibility of orthogonal multicolor
imaging. Fig. 8 shows the mixture of H6-MG and H10 surface-
displayed scFvs in the presence of MG-2p and OTB-SO3. Not only
is there negligible crossover, but little background fluorescence is
seen even without washing the unbound dye prior to imaging, once
again demonstrating the value of using fluorogenic dyes.8,11,13
Fluorescence microscopy
Fig. 7 shows confocal microscopy images of yeast surface-
expressed J10 in the presence of 200 nM OTB-SO3 and
DIR. Merged fluorescence and differential contrast images of
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Fig. 7 J10 cells imaged with 200 nM OTB-SO3 (left) and DIR (center), along with the overlaid images (right). Cells were not washed before imaging.
Samples were excited at 405 nm (OTB-SO3) and 561 nm (DIR).
fluorogenic dyes, simplifies the fluoromodule development process
by eliminating the need to (a) synthesize a biotinylated version of
the fluorogen and (b) magnetically pre-sort the library. Analogous
work has recently been conducted to improve the fluorescent
properties of a blue fluorescent protein by creating a library of
mutagenized chromophores and then screening the library using
flow cytometry.22
Four of our newly selected OTB-SO3-binding scFvs have
remarkably high quantum yields (ff > 0.8). These values compare
favorably with the quantum yields of Azurite (0.55), EBFP2
(0.56), and mTagBFP (0.63), which are some of the brightest
and most photostable proteins to date.22–24 These proteins feature
fluorophores that are covalently integrated into the protein struc-
ture. In addition, blue fluoromodules consisting of noncovalent
Fig. 8 H10 and H6-MG yeast cells with 200 nM OTB-SO3 and MG-2p.
dye-protein complexes were previously reported for trans-stilbene
fluorogens and antibody25 or scFv26 binding partners. Kd and
ff values ranged from 100–4000 nM and 0.1–0.8, respectively,
for those fluoromodules. The OTB-based fluoromodules reported
here exhibit generally higher affinity and similar or better quantum
yields than the stilbene variants.
The observation of enhanced fluorescence for OTB-SO3 in a vis-
cous glycerol solution indicates that a torsional motion contributes
to deactivation of the excited state in fluid solution, similar to other
unsymmetrical cyanine dyes as well as triphenylmethane dyes such
as malachite green.27,28 Twisting of the excited state dye about the
central methine bridge allows the dye to relax nonradiatively to the
ground state,◦provided the dye can twist beyond a critical torsional
angle (ca. 60 for thiazole orange).29 Restriction of this motion,
either by a bulk viscous solvent or by binding within a three-
dimensional pocket that constrains the torsional freedom of the
dye, blocks the nonradiative decay pathway, leading to significantly
enhanced fluorescence.
The Kd and ff data presented in Table 1 indicate that affinity
and fluorescence efficiency are not correlated. While all of the
fluoromodules show significantly higher fluorescence than for the
free dye, one of the highest affinity proteins, A6, exhibits the
lowest ff = 0.047. There are several possible explanations for this
observation. First, the dye could be bound by the benzoxazole
heterocycle, with few contacts to the benzothiazole ring system.
This would leave the benzothiazole half of the dye relatively
unconstrained and able to twist in the excited state, resulting
in weak fluorescence enhancement. (This would also account
Cells were not washed prior to imaging. Samples were excited at 405 nm
(OTB-SO3) and 633 nm (MG-2p).
Discussion
Our dye-scFv fluoromodules provide a new technology for mul-
ticolor fluorescent labeling and cellular imaging. A FAP binds
to a dye that is nonfluorescent when free in solution, resulting
in fluorogenic activation against a dark background of unbound
dye. The present work adds a new color to our toolbox of
fluoromodules, allowing for cellular labeling with a new blue
fluorescent dye, OTB-SO3.
In the selection method described in our previous reports,
the na¨ıve yeast library was initially subjected to two rounds of
magnetic sorting in which a biotinylated version of the fluorogenic
dye was immobilized on a magnetic bead coated with either strep-
tavidin or an anti-biotin antibody. The affinity-enriched library
was then sorted by FACS for yeast that activated fluorescence
from the dye.8,13 An important advance in our selection method
reported here involves bypassing of the magnetic selection steps,
i.e. the na¨ıve library was sorted directly by FACS for binding
to OTB-SO3.21 Since the library contains ca. 109 unique yeast,
only a quarter of the original library’s diversity was sorted over a
period of 6 h. Nevertheless, we selected at least six unique FAPs
in this experiment, indicating that there is sufficient diversity in
the library to allow selection of excellent (i.e. high affinity/high
quantum yield) binders after only partial screening of the library.
This approach, which we have since extended to several other
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for the failure of the t-butyl-modified analogue to bind to A6,
since the t-butyl group is attached to the benzoxazole ring.) A
second possibility is that the dye is bound to the protein in a
twisted conformation that is beyond the critical angle, leading
to a low quantum yield. While this would seem to require an
energetically unfavorable conformation for the dye, we note that
malachite green can bind to an RNA aptamer in a significantly
twisted conformation.28,30 A third explanation is that A6 has more
room in its binding pocket for torsional movement, resulting in
the bound dye losing energy through vibronic pathways. However,
this seems unlikely based on the emission spectra. The dye in A6
has a relatively narrow spectrum with a clear vibronic shoulder,
suggesting that the dye is tightly constrained, while in the very
bright scFv H10 it possesses the broadest emission spectrum. This
indicates that bright fluorescence can occur even in the presence of
some heterogeneity in the binding conformation and/or torsional
freedom, as long as the dye does not pass through its critical
torsional angle in the excited state.
The promiscuous binding ability of two of the clones was
especially interesting. A5 was able to bind t-butyl modified
versions of OTB but not to DIR, while J10 bound to DIR but
not to the t-butyl-OTB analogues. The other proteins exhibited
fluorogenic activation exclusively with OTB-SO3 and the unmod-
ified parent OTB. The range of dye-binding selectivities as well
as the lack of sequence homology exhibited by these proteins
indicates that the na¨ıve scFv library contains sufficient diversity
to provide numerous unique solutions to OTB-SO3 recognition.
High resolution structures determined by X-ray crystallography
or multidimensional NMR will aid in explaining the promiscuity
of these scFvs.
scFvs produced from cDNA. This library is also available from
Pacific Northwest National Laboratory. EBY100 yeast were used
to host the display library. The pPNL6 plasmids containing
individual clones were extracted from the yeast using a Zymoprep
I kit (Zymoresearch) and then transformed into Mach1TM-T1R
E. coli. Sequencing was conducted by GeneWiz. Expression of a
soluble protein version of the FAPs from E. coli was as described
previously.8
Fluorescence Activated Cell Sorting was conducted on a Becton
Dickinson FACSVantage SE with FACSDiva option. In the initial
selection round, 2.4 ¥ 108 cells, or ca. one fourth of the original
yeast library were sorted, a process which required approximately
6 h. Cells were incubated with unwashed 1 mM OTB-SO3 for 30 min
prior to sorting. Induction levels were measured using an Alexa
488 antibody binding to a c-myc epitope tag expressed alongside
the FAP, and the brightest 2% of cells in terms of OTB-SO3 and
induction signal were collected. Two additional selection rounds
were performed using 1 mM OTB-SO3, while in the last two rounds
the dye concentration was reduced to 100 nM to further increase
affinity. At the fifth selection, individual cells were sorted onto an
agar induction plate and allowed to grow. Individual colonies in
the presence of OTB-SO3 were examined on a Carl Zeiss LSM
510 Meta Confocal Microscope using a 405 nm laser and a 420-
480 nm bandpass filter; colonies with bright blue fluorescence were
selected for further analysis.
Optical spectroscopy
UV-Vis spectroscopy. UV-vis spectra were recorded on a
CARY-3Bio spectrophotometer. Sample temperatures were main-
◦
tained at 20 C using a thermoelectrically controlled Peltier cell
Finally, these fluoromodules are ready to be used for cell-
surface labeling applications. As shown in Fig. 8, the H10/OTB-
SO3 and H6-MG/MG-2p pairs are orthogonal and can be used
for simultaneous blue/red imaging of two different proteins.
Intracellular use will require engineering fluoromodules so that the
scFv component folds properly within the reducing environment
of the cytoplasm while also developing versions of the dyes that
are cell permeable.
holder.
Fluorescence quantum yields. Quantum yields for the OTB
dyes were determined using 9,10-diphenylanthracene in 100%
ethanol (ff = 0.95 at 20 ◦C) and quinine sulfate in 10% sulfuric
acid (ff = 0.55 at 25 ◦C) as calibration curve standards. Cresyl
violet in methanol (ff = 0.54 at 22 ◦C) and Cy5 in PBS (ff = 0.22 at
24 ◦C) were standards for the DIR measurements. Absorbance
measurements were conducted on a Cary 300 Bio UV-Visible
spectrophotometer, and fluorescence spectra were measured on
a Cary Eclipse fluorimeter at 20 ◦C. Quantum yields were
determined from samples having an absorbance of between 0.02
and 0.1 at and above lmax. A plot of absorbance versus the
integral of the fluorescence spectra was fit by linear regression,
giving a slope M. Quantum yields (Ux) were determined using the
following equation:
Conclusion
The results reported here show the development of a novel
family of dye-scFv fluoromodules based on a blue fluorogenic
cyanine dye, expanding the catalog of emission colors to the short
wavelength region of the visible spectrum.
Experimental
2
⎛
⎜
⎜
⎞⎛
M x hx
⎞
⎟
⎟
⎟
⎟
⎠
⎟
⎜
⎟
Fx = Fsd
⎜
⎟
⎜
⎜
⎝
⎜
⎜
sd
⎟
Materials
M
h
sd ⎠⎝
Synthesis and characterization of OTB dyes are described in sup-
porting information. PO-PRO-1 was purchased from Invitrogen
and used as received. DIR synthesis was described previously.19
Calf thymus DNA was purchased from Sigma–Aldrich and used
as received.
where Usd is the quantum yield of the standard dye, Mx and Msd are
the linear regression slopes of the sample and standard as defined
previously, and hx and hsd are the refractive indices. Refractive
indices of 1.333 for water (buffer), 1.3614 for ethanol, 1.0661 for
10% sulfuric acid, and 1.4583 for glycerol were used.
Fluorescence enhancements. Fluorescence enhancement val-
ues were calculated based on the fluorescence ratio for 200 nM
dye bound to protein versus dye in buffer. These values were not
corrected for differences in absorbance in the two samples.
Selection of yeast surface-displayed clones that activate OTB-SO3
The na¨ıve yeast cell surface-display library was provided by Dane
Wittrup of MIT and consists of ca. 8 ¥ 108 recombinant human
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Org. Biomol. Chem., 2011, 9, 1012–1020 | 1019
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Acknowledgements
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K.J.Z. gratefully acknowledges support from a DoD, Air Force
Office of Scientific Research, NDSEG Fellowship 32 CFR 168a.
This work is supported by the U.S. National Institutes of Health
(grant U54 RR022241). NMR instrumentation at CMU was
partially supported by NSF (CHE-0130903). Mass spectrometers
were funded by NSF (DBI-9729351). MG-2p was a generous
gift from Brigitte Schmidt. We thank Drs. Gayathri Withers and
Roberto Gil for expert assistance with NMR characterization of
dyes. We are grateful to Dane Wittrup for providing an aliquot of
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