вход по аккаунту


Colorimetric Screening of DNA-Binding Molecules with Gold Nanoparticle Probes.

код для вставкиСкачать
Colorimetric Sensors
DOI: 10.1002/ange.200504277
Colorimetric Screening of DNA-Binding
Molecules with Gold Nanoparticle Probes**
Min Su Han, Abigail K. R. Lytton-Jean, ByungKeun Oh, Jungseok Heo, and Chad A. Mirkin*
Combinatorial chemistry is a powerful tool that enables
scientists to synthesize many compounds within a short time
period.[1] This ability to synthesize large libraries of compounds has enabled the development of many potential
anticancer drugs.[2] However, one of the bottlenecks in drug
discovery is the selection of drug candidates from the many
compounds within these libraries.[3] To overcome this problem, high-throughput screening methods are used to screen
large libraries of potential drug candidates for biological
activity.[4] Many anticancer drugs, such as doxorubicine,
daunorubicin, and amsacrine, are known to interact reversibly
with DNA to form a drug/DNA complex.[5] Generally, the
strength of binding between the anticancer drug and DNA
correlates with the biological activity of the drug and is
therefore an important piece of information in the screening
process.[6] In the past, typical screening processes have
included mass spectrometry, nuclear magnetic resonance
(NMR) spectroscopy, light scattering, and electrochemistry.[7]
Unfortunately, these methods are not applicable to highthroughput screening. Fluorescence screening compatible
with high-throughput protocols have been developed only
Herein, we describe a colorimetric assay for determining
the binding affinities between potential DNA-binding molecules and duplex DNA by using networks of Au nanoparticles
interconnected with duplex DNA.[9] The colorimetric readout
can be visualized with the naked eye without requiring
additional instrumentation. DNA-functionalized Au nanoparticles have been used previously to detect DNA, proteins,
and metal ions.[10] To the best of our knowledge, this is the first
example where DNA-functionalized Au nanoparticles are
used to determine relative binding affinities of molecules to
duplex DNA. This capability is applicable to screening
libraries for drug candidates in a high-throughput fashion.
This method utilizes the aggregation-induced, red-to-blue
[*] M. S. Han, A. K. R. Lytton-Jean, B.-K. Oh, J. Heo, Prof. C. A. Mirkin
Department of Chemistry and
The Institute for Nanotechnology
2145 Sheridan Road, Evanston, IL, 60208-3113 (USA)
Fax: (+ 1) 847-467-5123
[**] This work was supported by the NIH through a Director’s Pioneer
Award to C.A.M., the NCI through a CCNE, the NSF, and the
AFOSR. M.S.H. is grateful for a fellowship from the Korea Research
Foundation (MOEHRD, Basic Research Promotion Fund) (Grant
no. M01-2003-000-10140-0).
Supporting information for this article is available on the WWW
under or from the author.
Angew. Chem. 2006, 118, 1839 –1842
2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
color change associated with
13-nm Au nanoparticles.
Gold nanoparticles were
functionalized with one of
two complementary thiolmodified
strands and are denoted
NP-1 and NP-2. When NP-1
and NP-2 are combined, they
form aggregates through a
reversible DNA-hybridization process. This process
results in a red-to-blue color
change caused by a red-shifting and dampening of the
nanoparticle plasmon resonance.[9] Increasing the temperature above the melting
temperature (Tm) of the
DNA reverses the process,
and the particles dissociate
with a concomitant blue-toScheme 1. Schematic representation of the structural and color change of nanoparticle/DNA-bindingred color change. The meltmolecule assemblies at a specific temperature (T1).
ing transition occurs over a
very narrow temperature
range with the first derivative of the transition exhibiting a
full width at half maximum (fwhm) of 1–2 8C. This transition is
significantly more narrow than the transition associated with
a nanoparticle-free duplex of identical length and sequence
(fwhm 10–12 8C)[10a] and is characteristic of nanoparticle
and polymer probes heavily functionalized with oligonucleotide strands.[11] When NP-1 and NP-2 are combined in the
presence of any of the known duplex DNA-binding molecules
in Table 1, duplexes form between the Au nanoparticles that
Table 1: Melting temperatures (Tm) of nanoassemblies and duplex DNA
in the presence of DNA-binding molecules.
Tm[a] [8C]
Tm[b] [8C]
no DNA binder
[a] Conditions: NP-1 and NP-2 (each 1.5 nm) in sodium phosphate
buffer (10 mm; pH 7.0) containing sodium chloride (100 mm). [b] Conditions: DNA-1 and DNA-2 (each 2.0 mm) in sodium phosphate buffer
(10 mm; pH 7.0) containing sodium chloride (300 mm).
are more stable than those in the absence of the DNA-binding
molecules (Scheme 1). This increased stability is reflected by
an increase in the melting temperature. Note that the shape
and width of the transition is almost independent of the DNAbinding molecule (Figure 1). Therefore, by monitoring the
blue-to-red color transition of hybridized oligonucleotidemodified Au nanoparticles in the presence of different
Figure 1. Melting curves of A) DNA-1 and DNA-2 (no nanoparticles)
and B) NP-1 and NP-2 assemblies in the absence (h) and presence of
DNA-binding molecules DAPI (a), DNR (b), EB (c), 9-AA (d), EIPT (e),
AQ2A (f), and AMSA (g).
compounds, we can determine the relative binding strength
of DNA-binding molecules with the naked eye. It is assumed
that any effect on DNA-binding affinity caused by increased
temperature will be similar for all DNA-binding molecules
because of the similar DNA-binding interactions (p–p stack-
2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. 2006, 118, 1839 –1842
ing, electrostatic, and hydrogen bonding) between DNA
binders and duplex DNA.
The assay is initiated by mixing NP-1 and NP-2 (each at
1.5 nm) in a 1:1 molar ratio in 10 mm phosphate-buffered
saline (PBS) buffer (0.1m NaCl; pH 7.0). The melting temperatures of the nanoassemblies (NP-1 and NP-2) were then
determined in the presence of 4’,6-diamidino-2-phenylindole
(DAPI), ellipticine (EIPT), amsacrine (AMSA), daunorubicin (DNR), anthraquinone-2-carboxylic acid (AQ2 A), ethidium bromide (EB), and 9-aminoacridine (9-AA) (5 mm),
respectively. UV/Vis melting profiles were measured by
monitoring the absorbance at 520 nm with increasing temperature (scan rate 0.5 8C min 1). The melting temperatures were
determined by taking the maxima of the first derivative of the
melting curves. Control experiments were run by determining
the melting temperatures of unmodified duplex DNA at
260 nm (DNA-1: 5’-TAACAATAA-3’, DNA-2: 5’TTATTG TTA-3’) in the presence of all seven DNA-binding
molecules (Table 1).
The strength of binding between anticancer drugs and
DNA generally correlates with the biological activity of the
drug [6] and is reflected in an increased melting temperature
for the DNA.[12] Therefore, we can screen for the relative
binding affinities of molecules for a particular DNA sequence
that links the nanoparticle assembly by monitoring the color
change as a function of temperature with all other conditions
kept constant. The trends in the melting temperatures of the
nanoparticles and the unmodified control duplex (with and
without DNA-binding molecules) are similar (Table 1 and
Figure 1). The addition of a known binder results in an
enhanced Tm value. The Tm values depend on many factors,
including salt concentration, DNA concentration, and binding
affinity of the particle for complementary DNA. Indeed, in
this regard, the nanoparticle assemblies provide several
substantial advantages over direct detection of DNA binding
by UV/Vis spectroscopy. The melting transition for the DNAinterlinked assemblies occurs over a very narrow temperature
range compared to the broad melting transitions of nanoparticle-free duplex DNA (Figure 1 A). This allows a more
precise analysis of the change in melting temperature of the
nanoparticle assemblies (Figure 1 B). Furthermore, the presence of the DNA-binding molecule induces a more substantial temperature increase in the nanoparticle system relative
to the nanoparticle-free duplex system (Table 1), which
allows us to identify and differentiate between weak-,
intermediate-, and strong-DNA-binding molecules more
easily. For example, the Tm value monitored by UV/Vis
spectroscopy is 19.1 8C for duplex DNA without DAPI and
27.8 8C in the presence of DAPI (D = 8.7 8C). However, the
Tm value of the same sequences attached to nanoparticle
assemblies shifts from 26.4 to 50.4 8C (D = 24 8C) upon
introduction of DAPI. The dramatic increase in the melting
temperature of the nanoparticle aggregates in the presence of
DNA-binding molecules is due, in part, to an extremely high
ratio of DNA-binding molecules to nanoparticle DNA
(Figure 1 B, ratio = 33:1) relative to the nanoparticle-free
case (Figure 1 A, ratio = 5:2). Such a high ratio is possible
because the nanoparticles absorb light much more strongly
(at 260 and 520 nm) than the DNA, which allows us to work at
Angew. Chem. 2006, 118, 1839 –1842
very low concentrations of nanoparticle probe while maintaining a high concentration of DNA-binding molecules. It is
impossible to work at this high DNA-binding-molecule/DNA
ratio when using nanoparticle-free DNA because many
DNA-binding molecules absorb light at the same wavelength
as DNA and thus interfere with the signal of the oligonucleotides at 260 nm. Also, the absorbance of some DNA-binding
molecules at 260 nm is sensitive to environmental changes
(temperature, binding, dielectric) and interferes with melting
measurements made by monitoring the absorbance of DNA
at 260 nm.[13] Note that melting transitions for the nanoparticle aggregates were monitored at 520 nm (corresponding
to the gold plasmon resonance) and those for duplex DNA
were monitored at 260 nm.
In general, assay methods that can detect drug candidates
with the naked eye without resorting to any instrumentation
are convenient, and, for this reason, an assay that could screen
for drug candidates on the basis of DNA-binding strength
would be of great interest. At present, there are no
colorimetric assays that provide this capability. The use of
DNA-functionalized Au nanoparticles for this purpose is
demonstrated in Figure 2. As the temperature increases, the
Figure 2. The color change of the nanoassembly (NP-1 and NP-2, each
1.5 nm) in the absence (CON) and presence of DNA-binding molecules (5 mm) specific temperatures.
color changes from blue to red at specific temperatures. All
eight cells (one control and seven DNA-binding molecules)
appear light blue/purple at 25 8C. At 30 8C, the nanoassemblies containing the control and weak-DNA-binding molecules turn red; only the nanoassemblies containing DAPI,
DNR, and EB remain blue/purple. Increasing the temperature to 40 8C causes all of the samples except the nanoassembly containing DAPI—a strong-DNA-binding molecule—to turn red. These results show the discrimination
between weak, intermediate, and strong-DNA-binding molecules by an easily identified color change. The trend of the
binding affinities for DNA was determined to be DAPI >
DNR EB > other DNA-binding molecules, which is consistent with the control experiments involving serial analysis of
each DNA-binding molecule with nanoparticle-free duplex
In summary, we have developed a colorimetric assay for
determining the relative binding strengths of molecules to
duplex DNA by monitoring the color of nanoparticle network
2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
materials exposed to the DNA binders with increasing
temperature. The assay has a number of advantages over
conventional systems that monitor molecule–DNA interactions directly or with molecular fluorophore probes. The
sharp melting transition associated with network materials
made from DNA-interlinked gold nanoparticles, their strong
absorbance at 520 nm, and the large perturbations in the
Tm values upon analyte binding allow significantly better
discrimination between weak, intermediate, and strongDNA-binding molecules. Furthermore, the simplicity of this
assay should make it more convenient than other methods
that rely on mass spectrometry, NMR spectroscopy, light
scattering, electrochemistry, and fluorometry. Finally, this
assay can be adapted easily to high-throughput screening
methods, which can be used to determine potential anticancer
agents from large combinatorial libraries on the basis of their
duplex DNA-binding affinity.
Shlyahovsky, I. Willner, J. Am. Chem. Soc. 2004, 126, 11 768 –
11 769; f) J. Liu, Y. Lu, J. Am. Chem. Soc. 2003, 125, 6642 – 6643.
[11] K. J. Watson, S.-J. Park, J.-H. Im, S. T. Nguyen, C. A. Mirkin, J.
Am. Chem. Soc. 2001, 123, 5592 – 5593.
[12] J. Joseph, E. Kuruvilla, A. T. Achuthan, D. Ramaiah, G. B.
Schuster, Bioconjugate Chem. 2004, 15, 1230 – 1235.
[13] S. Tanaka, Y. Baba, A. Kagemoto, Makromol. Chem. 1981, 182,
1475 – 1480.
Received: December 1, 2005
Published online: February 15, 2006
Keywords: DNA · gold · high-throughput screening ·
nanostructures · sensors
[1] a) L. A. Thompson, J. A. Ellman, Chem. Rev. 1996, 96, 555 – 600;
b) F. Balkenhohl, C. von dem Bussche-HEnnefeld, A. Lansky, C.
Zechel, Angew. Chem. 1996, 108, 2436 – 2487; Angew. Chem. Int.
Ed. Engl. 1996, 35, 2288 – 2337.
[2] a) D. L. Boger, B. E. Fink, M. P. Hedrick, J. Am. Chem. Soc.
2000, 122, 6382 – 6394; b) V. Nesterenko, K. S. Putt, P. J. Hergenrother, J. Am. Chem. Soc. 2003, 125, 14 672 – 14 673.
[3] M. J. A. Walker, T. Barrett, L. J. Guppy, Targets 2004, 3, 208 –
[4] a) P. A. Johnston, P. A. Johnston, Drug Discovery Today 2002, 7,
353 – 363; b) D. L. Boger, J. Desharnais, K. Capps, Angew. Chem.
2003, 115, 4270 – 4309; Angew. Chem. Int. Ed. 2003, 42, 4138 –
4176; c) S. Wang, T. B. Sim, Y.-S. Kim, Y.-T. Chang, Curr. Opin.
Chem. Biol. 2004, 8, 371 – 377.
[5] a) X.-L. Yang, A. H.-J. Wang, Pharm. Therap. 1999, 83, 181 –
215; b) C. Bailly, W. A. Denny, L. E. Mellor, L. P. G. Wakelin,
M. J. Waring, Biochemistry 1992, 31, 3514 – 3524.
[6] a) D. Řeha, M. KabelKč, F. RyjKček, J. Šponer, J. E. Šponer, M.
Elstner, S. Suhai, P. Hobza, J. Am. Chem. Soc. 2002, 124, 3366 –
3376; b) I. Antonini, P. Polucci, T. C. Jenkins, L. R. Kelland, E.
Menta, N. Pescalli, B. Stefanska, J. Mazerski, S. Martelli, J. Med.
Chem. 1997, 40, 3749 – 3755.
[7] a) S. A. Hofstadler, R. H. Griffey, Chem. Rev. 2001, 101, 377 –
390; b) H. Robinson, W. Priebe, J. B. Chaires, A. H.-J. Wang,
Biochemistry 1997, 36, 8663 – 8670; c) R. F. Pasternack, C.
Bustamante, P. J. Collings, A. Giannetto, E. J. Gibbs, J. Am.
Chem. Soc. 1993, 115, 5393 – 5399; d) C.-Z. Li, Y. Liu, H. T.
Luong, Anal. Chem. 2005, 77, 478 – 485.
[8] D. L. Boger, B. E. Fink, S. R. Brunette, W. C. Tse, M. P. Hedrick,
J. Am. Chem. Soc. 2001, 123, 5878 – 5891.
[9] C. A. Mirkin, R. L. Letsinger, R. C. Mucic, J. J. Storhoff, Nature
1996, 382, 607 – 609.
[10] a) R. Elghanian, J. J. Storhoff, R. C. Mucic, R. L. Letsinger,
C. A. Mirkin, Science 1997, 277, 1078 – 1080; b) L. He, M. D.
Musick, S. R. Nicewarner, F. G. Salinas, S. J. Benkovic, M. J.
Natan, C. D. Keating, J. Am. Chem. Soc. 2000, 122, 9071 – 9077;
c) D. J. Maxwell, J. R. Taylor, S. Nie, J. Am. Chem. Soc. 2002,
124, 9606 – 9612; d) T. Niazov, V. Pavlov, Y. Xiao, R. Gill, I.
Willner, Nano Lett. 2004, 4, 1683 – 1687; e) V. Pavlov, Y. Xiao, B.
2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. 2006, 118, 1839 –1842
Без категории
Размер файла
315 Кб
screening, molecules, colorimetry, dna, gold, probes, binding, nanoparticles
Пожаловаться на содержимое документа