Molecular tripods showing fluorescence enhancement upon binding to streptavidin

Article abstract of DOI:10.1021/ol047801h

(Chemical Equation Presented) We introduce a new approach to biotin-streptavidin assays based on a molecular tripod which consists of biotin, a fluorophore, and a quencher. The interaction between streptavidin and molecular tripods perturbs the ground-state quencher-fluorophore dimeric conformation in the absence of streptavidin and diminishes the intrinsic self-quenching of a quencher-fluorophore pair. The emission intensity of the molecular tripods plus streptavidin is 3.5-5.2 times that of molecular tripods in the absence of streptavidin.

Full text of DOI:10.1021/ol047801h

ORGANIC
LETTERS
2005
Vol. 7, No. 1
111-114
Molecular Tripods Showing
Fluorescence Enhancement upon
Binding to Streptavidin
Tae Woo Kim,† Hey Young Yoon,† Jung-hyun Park,† Oh-Hoon Kwon,†
Du-Jeon Jang,*,† and Jong-In Hong*,†,‡
Department of Chemistry, College of Natural Sciences, Seoul National UniVersity,
Seoul 151-747, Korea, and Center for Molecular Design and Synthesis, KAIST,
Daejon 305-701, Korea
jihong@.plaza.snu.ac.kr
Received October 26, 2004
ABSTRACT
We introduce a new approach to biotin
quencher. The interaction between streptavidin and molecular tripods perturbs the ground-state quencher
in the absence of streptavidin and diminishes the intrinsic self-quenching of a quencher fluorophore pair. The emission intensity of the
molecular tripods plus streptavidin is 3.5 5.2 times that of molecular tripods in the absence of streptavidin.
−streptavidin assays based on a molecular tripod which consists of biotin, a fluorophore, and a
−fluorophore dimeric conformation
−
−
Since biotin shows extremely strong binding affinity toward
avidin and streptavidin, the noncovalent binding interaction
between biotin and (strept)avidin has found widespread
bioanalytical applications such as detection of biomolecules,
clinical diagnosis, and immunoassay.1 Therefore, specific
measurement of (strept)avidin is often needed. For the
biotin-(strept)avidin assays, three kinds of techniques such
as radioligand binding, enzyme assay, and photometrics/
fluorimetrics have been developed.2 Of these, optical and
fluorescence methods offer significant advantages for ana-
lytical purposes in sensitivity and simplicity.3 The simplest
approach to the biotin-(strept)avidin monitoring would be
using biotin-fluorophore conjugates. However, most biotin-
fluorophore conjugates suffer from fluorescence diminish-
ment upon binding to (strept)avidin presumably due to
resonance energy transfer,4 except for biotin-fluorophore
conjugates with long spacers.5 Therefore, it would be useful
(2) (a) Groman, E. V.; Rothenberg, J. M.; Bayer, E. A.; Wilchek, M.
Methods Enzymol. 1990, 184, 217. (b) Barbarakis, M. S.; Qaisi, W. G.;
Daunert, S.; Bachas, L. G. Anal. Chem. 1993, 65, 457. (c) Mock, D. M.;
Horowitz, P. Methods Enzymol. 1990, 184, 234. (d) D’Auria, S.; Lakowicz,
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Life Sci. 2003, 74, 489.
(3) (a) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 2nd
ed.; Kluwer Academic and Plenum Publishers: New York, 1999. (b) de
Silva, A. P.; Gunaratne, H. Q. N.; Gunnlaugsson, T.; Huxley, A. J. M.;
McCoy, C. P.; Rademacher, J. T.; Rice, T. E. Chem. ReV. 1997, 97, 1515.
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Acta 1999, 1427, 44.
† Seoul National University.
‡ KAIST.
(1) (a) Wilchek, M.; Bayer, E. A. Methods of Enzymology; Academic
Press: San Diego, 1990; Vol. 184. (b) Christopoulos, T. K.; Diamandis, E.
P. Immunoassay; Academic Press: San Diego, CA, 1996; Vol. 184. (c)
Hermanson, G. T. Bioconjugate Techniques; Academic Press: San Diego,
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10.1021/ol047801h CCC: $30.25
© 2005 American Chemical Society
Published on Web 12/15/2004

Figure 1. Schematic drawing for the off-on fluorescence mech-
anism of a molecular tripod in streptavidin binding.
to develop fluorescent probes showing fluorescence enhance-
ment upon binding to (strept)avidin.6 Recently, Lo and co-
workers developed rhenium(I) polypyridine-biotin com-
plexes that show luminescence enhancement and lifetime
elongation upon binding to avidin.6a.b Sleiman recently
showed that ruthenium(II) phenanthroline-biotin conjugates
can be used as luminescent probes for avidin with lumines-
cence enhancement when binding to avidin.6e We were
interested in the development of molecular tripod (biotin-
quencher-fluorophore conjugate)-based streptavidin probes
with novel off-on fluorescence mechanisms.
Here we report on new fluorescent probes that are useful
in the detection of streptavidin in homogeneous solution. The
probes are composed of biotin, a fluorophore, a quencher,
and a spacer. The quencher-fluorophore pair as a reporting
group can monitor any fluorescence change before and after
the biotin-streptavidin binding event.7,8 The spacer maintains
a quencher-fluorophore pair in spatial proximity, causing
the fluorescence of the pair to be quenched in an aqueous,
unbound state via the ground-state intramolecular complex
between the fluorophore and the quencher.9 When the probe
encounters streptavidin, it forms a streptavidin-biotin com-
plex that perturbs the ground-state intramolecular complex
and eventually impedes the self-quenching of a quencher-
fluorophore pair (Figure 1). We call these probes “molecular
Figure 2. Nomenclature of the molecular tripods and reference
compounds used in this work.
tripods” because they have trifurcated structures. Because
streptavidin binding triggers fluorescence enhancement, in
principle, molecular tripods can be used for the detection of
streptavidin in homogeneous assay.
The molecular tripods consist of biotin as a substrate for
streptavidin, fluorescein, coumarin 343, and rhodamine as
fluorophores and carbazole as a quencher (Figure 2). A
spacer based on 5-hydroxyisophthalic acid was used for ease
of synthesis. Streptavidin, a nonglycosylated 52 800 Da
protein with a near-neutral isoelectric point, binds four biotins
per molecule with high affinity and selectivity. We also
synthesized two kinds of reference compounds; quencher-
fluorophore conjugates (QF, QC, QR) and biotin-fluoro-
phore conjugates (BF, BC, BR) (Figure 2).10,11 In the case
of the biotin-fluorophore conjugates, biotin was directly
connected to fluorophores without the 5-hydroxyisophthalic
acid spacer.10 To define the quenching efficiency, we also
prepared alkyl derivatives of fluorophores (F′, C′, R′).10
To ensure the spontaneous formation of intramolecular
ground-state dimeric complexes in the quencher-fluorophore
pairs, self-quenching efficiency was measured for carbazole-
containing probes (Table 1). Because the protein surface
usually works as a fluorescence energy transfer sinker,4,11,12
(5) (a) Gruber, H. J.; Marek, M.; Schindler, H.; Kaiser, K. Bioconjugate
Chem. 1997, 8, 552. (b) Marek, M.; Kaiser, K.; Gruber, H. J. Bioconjugate
Chem. 1997, 8, 560. (c) Gruber, H. J.; Hahn, C. D.; Kada, G.; Riener. C.
K.; Harms, G. S.; Ahrer, W.; Dax, T. G.; Knaus, H.-G. Bioconjugate Chem.
2000 11, 696.
(6) (a) Lo, K. K.-W.; Hui, W.-K.; Ng, D. C.-M. J. Am. Chem. Soc. 2002,
124, 9344. (b) Lo, K. K.-W.; Tsang, K. H.-K. Organometallics 2004, 23,
3062. (c) Lo, K. K.-W.; Chan, J. S.-W.; Lui, L.-H.; Chung, C.-K.
Organometallics 2004, 23, 3108. (d) Lo, K. K.-W.; Lee, T. K.-M. Inorg.
Chem. 2004, 43, 5275. (e) Slim, M.; Sleiman, H. F. Bioconjugate Chem.
2004, 15, 949.
(7) Neuweiler, H.; Schulz, A.; Vaiana, A. C.; Smith, J. C.; Kaul, S.;
Wolfrum, J.; Sauer, M. Angew. Chem., Int. Ed. 2002, 41, 4769.
(8) Quencher-fluorophore pairs have been applied for sensing small
molecules to high molecular weight analytes. (a) For cations: Aoki, I.; Sakai,
T.; Shinkai, S. J. Chem. Soc., Chem. Commun. 1992, 730. (b) For
peptides: Chen, C.-T.; Wagner, H.; Still, W. C. Science 1998, 279, 851.
(c) For protein-ligand binding: Wei, A.-P.; Blumenthal, D. K.; Herron, J.
N. Anal. Chem. 1994, 66, 1500. (d) For cholic acid: Hossain, M. A.;
Hamasaki, K.; Takahashi, K.; Mihara, H.; Ueno, A. J. Am. Chem. Soc.
2001, 123, 7435.
(9) (a) Packard, B. Z.; Toptygin, D. D.; Komoriya, A.; Brand, L. Proc.
Natl. Acad. Sci. U.S.A. 1996, 93, 11640. (b) Takakusa, H.; Kikuchi, K.;
Urano, Y.; Higuchi, T.; Nagano, T. Anal. Chem. 2001, 73, 939. (c)
Johansson, M. K.; Fidder, H.; Dick, D.; Cook, R. M. J. Am. Chem. Soc.
2002, 124, 6950. (d) Johansson, M. K.; Cook, R. M. Chem. Eur. J. 2003,
9, 3466.
(10) See the Supporting Information.
(11) Specific binding of biotin-fluorescein conjugates towards strepta-
vidin was accompanied by fluorescence quenching: see ref 4.
(12) Gruber, H. J.; Kada, G.; Marek, M.; Kaiser, K. Biochim. Biopys.
Acta 1998, 1381, 203.
112
Org. Lett., Vol. 7, No. 1, 2005

Table 1. Quenching Efficiencya,b (QE) and Enhancement Factorc (EF)
A. fluorescein basedd B. coumarin 343 basede
EF QE EF
BF + S4,11,12
BQC + S
3.5
C. rhodamine basdf
EF
BQR + S
5.2
QE
QE
QF
BQF
BQF + S
QC
BQC
BC + S
QR
BQR
BR + S
-99%
-97%
0.20
-98%
-99%
0.30
-99%
-99%
1.05
a Buffer A (100 mM NaCl, 50 mM NaH2PO4, 1 mM EDTA, pH adjusted to 7.5 with NaOH) was used. b QE ) {[FI - FI0]/FI0} × 100, FI0 ) fluorescence
intensity (FI) of free fluorophore X (F for A, C for B, and R for C) at 50 nM, FI ) fluorescence intensity of the probe at 50 nM. c EF ) FIwith/FIwithout
,
FIwithout ) fluorescence intensity of the probe without streptavidin, FIwith ) fluorescence intensity of the probe with streptavidin (15 nM) at [probe] ) 50 nM.
Values of EF greater than 1 indicate the fluorescence enhancement by streptavidin addition and values less than 1 the fluorescence quenching by streptavidin
addition; d λex ) 490 nm, λem
) 515 nm. e λex ) 446 nm, λem
) 500 nm. f λex ) 543 nm, λem
) 570 nm. S ) streptavidin.
max
max
max
we also have to check the fluorescence change in biotin-
fluorophore conjugates (BF, BC, BR) after streptavidin
binding. The high quenching efficiency (≈ 97-99%) indi-
cates that the designed molecules (BQX, QX, X ) F, C, R)
form ground-state intramolecular dimers over the 5-hydroxy-
isophthalic acid spacer. In the case of BQC and BQR, the
quenched fluorescence of the molecular tripods was partially
recovered after streptavidin binding. The emission intensity
of the BQC + streptavidin and BQR + streptavidin is 3.5
and 5.2 times that of BQC and BQR in the absence of
streptavidin, respectively (Table 1). However, the fluores-
cence of BQF decreased after streptavidin binding. This
result implies that we have to consider the interaction
between the surface of streptavidin around the binding pocket
and a fluorophore in order to successfully design the
molecular tripods.
biotin-fluorescein conjugate with a long 14-atom spacer
possessed several drawbacks such as long assay times and
moderate sensitivity, resulting from the anti-cooperative
binding of streptavidin.12 The sigmoid titration curve in the
inset of Figure 3 reflects the 1:4 (streptavidin/biotin) stoi-
chiometry and anti-cooperative binding. Therefore, we were
not able to determine Kd from the curve. Nevertheless, the
saturation pattern above 10 nM is consistent with the known
binding property of streptavidin.
To prove that the molecular tripod is a selective probe for
streptavidin, we measured the fluorescence enhancement of
BQC and BQR caused by the addition of streptavidin and
bovine serum albumin (BSA) under the same conditions
(Figure 4 and Supporting Information). While addition of
The fluorescence intensity of BC was reduced by increas-
ing streptavidin, while the response of BQC was opposite
(Table 1, Figure 3). As shown in Figure 3, an enhancement
Figure 4. Fluorescence intensity changes of BQR by the addition
of streptavidin, BSA, and both. [BQR] ) 50 nM in buffer A;
[streptavidin] ) 400 ng mL-1, [BSA] ) 400 ng mL-1, [streptavidin
+ BSA] ) 400 ng mL-1 for streptavidin and 400 ng mL-1 for
BSA.
Figure 3. Fluorescence responses of 50 nM BQC to streptavidin
addition (9 ) 0 nM, b ) 15 nM). The inset shows the continuous
fluorescence titration of BQC (50 nM) with streptavidin.
BSA to each tripod caused little change in the fluorescence
intensity, streptavidin addition resulted in larger emission
enhancement. In particular, the fluorescence intensity changes
little upon addition of BQR to the mixture of streptavidin
and BSA. The results imply that the molecular tripod
functions as a specific streptavidin probe.
The effect of added biotin in the emission spectrum of
BQR-streptavidin complex is shown in Figure 5. Addition
of increasing amounts of biotin to the preformed complex
of BQR-streptavidin in more than a 4:1 ratio resulted in
of 3.5 was observed in the presence of streptavidin. The
results show that our strategy, namely the diminution of
fluorescence self-quenching by streptavidin-biotin binding,
works well in the molecular tripod.
For real-time monitoring, the time dependence of the
BQC-streptavidin interaction was checked. It turns out that
the fluorescence intensity stabilizes after a short period of
vortexing and the value was hardly changed even after 1
day. It was known that the fluorimetric assay involving the
Org. Lett., Vol. 7, No. 1, 2005
113

A qualitative prospect for the interaction between the
streptavidin surface and a tripod can be obtained from X-ray
crystal structure (PDB ID 1STP).13 Lys-121 of streptavidin-
biotin complex is placed within 15 Å from the carboxy
terminal of biotin. The positive charge around the streptavidin
binding pocket gives a clue to understanding why coumarin
343 and rhodamine work for molecular tripods and why
fluorescein does not. A net negatively charged, fluorescein-
based probe may undergo electrostatic attraction with Lys-
121, so the time/space proximity between the protein surface
and the fluorescein-based probe makes the protein surface
function as a fluorescence sinker. Compared with a fluorescein-
based one, neutral coumarin 343-based, and net positively
charged rhodamine-based probes have no attractive interac-
tion with Lys-121. Although this explanation may be too
simplified, this rough approach suggests a guideline for
molecular tripod design. From the protein surface analysis,
we can rule out fluorophores which might interact with hot
residues around the protein-substrate binding pocket.
In summary, we have developed a new biotin-streptavidin
assay technique using a molecular tripod based on a biotin-
streptavidin interaction. The interaction between streptavidin
and the molecular tripod perturbs ground-state quencher-
fluorophore dimeric conformation and diminishes the intrin-
sic self-quenching of the quencher-fluorophore pair. The
fluorescence enhancement brought about by streptavidin
binding is comparable to the fluorescence quenching of
biotin-4-fluorescein.4 Positive fluorescence response, rapid
equilibrium, saturated titration behavior, and streptavidin
specificity show that the molecular tripod is suitable for
detecting streptavidin in homogeneous solutions. This new
approach is potentially useful for the study of various
protein-substrate interactions.
Figure 5. Fluorescence changes of BQR (500 nM) in the presence
of streptavidin (100 nM) in buffer A solution with the addition of
biotin solution (0-1000 nM). After each mixture of BQR and
streptavidin was equilibrated for 1 h, a different amount of biotin
was added and the mixture equilibrated for 20 h. Maximum
emission intensities at 570 nm were recorded after each addition
of biotin solution. The emission intensity of the free BQR (500
nM) is about 40 au.
gradual decrease in the fluorescence intensity, until an
approximately 4:1 ratio of biotin/streptavidin was reached,
upon which the fluorescence intensity of the tripod did not
change even with further addition of biotin. This result
indicates that BQR is displaced from streptavidin by biotin.
Although biotin itself binds more strongly to streptavidin
than BQR, BQR still strongly binds to streptavidin with
fluorescence enhancement.
The fluorescence lifetime measurements show a small
lifetime increase for molecular tripods (+5% for BQC, +8%
for BQR) and some decrease for biotin-fluorophore con-
jugates (-19% for BC, -11% for BR) upon binding to
streptavidin. However, the lifetime changes are too small to
explain the fluorescence enhancement by energy transfer in
an excitation state. Absorption spectrum of BQR shows that
the peak at shorter wavelengths (about 520 nm) decreases
after the addition of streptavidin (see the Supporting Infor-
mation). The change in shape of the absorption spectrum
indicates that the population of H-type ground-state intramo-
lecular dimers decreases after streptavidin binding. The
fluorescence quenching of an unbound probe results from
the H-type ground-state intramolecular complex.9 The con-
gested environment and protein surface interaction around
the protein binding pocket in the bound state disrupts the
H-type ground-state intramolecular structure of the unbound
state, and thus, the quenched fluorescence is recovered.
Acknowledgment. Financial support from the Basic
Research Program of the KOSEF (Grant No. R02-2003-000-
10055-0) is gratefully acknowledged. T.W.K., J.-h.P., and
H.Y.Y. thank the Ministry of Education for the award of
the BK 21 fellowship. D.-J.J. is thankful for the Strategic
National R&D Program.
Supporting Information Available: Synthetic scheme
and spectral data of the molecular tripods and reference
compounds in Figure 2, fluorescence enhancement factor by
the addition of streptavidin, BSA, and both, and absorption
spectra of BQR and BQR + streptavidin. This material is
available free of charge via the Internet at http://pubs.acs.org.
OL047801H
(13) Weber, P. C.; Ohlendorf, D. H.; Wendoloski, J. J.; Salemme, F. R.
Science 1989, 243, 85.
114
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