Studying forkhead box protein A1–DNA interaction and ligand inhibition using gold nanoparticles, electrophoretic mobility shift assay, and fluorescence anisotropy

Analytical Biochemistry 448 (2014) 95–104

Contents lists available at ScienceDirect

Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio

Studying forkhead box protein A1–DNA interaction and ligand inhibition using gold nanoparticles, electrophoretic mobility shift assay, and fluorescence anisotropy Khin Moh Moh Aung a, Siu Yee New a, Shuzhen Hong b, Laura Sutarlie a, Michelle Gek Liang Lim b, Si Kee Tan b, Edwin Cheung b,c,d,⇑, Xiaodi Su a,⇑ a

Institute of Materials Research and Engineering, Agency for Science, Technology, and Research (A*STAR), Singapore Cancer Biology and Pharmacology, Genome Institute of Singapore, Agency for Science, Technology, and Research (A*STAR), Singapore c Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore d School of Biological Sciences, Nanyang Technological University, Singapore b

a r t i c l e

i n f o

Article history: Received 28 June 2013 Received in revised form 11 November 2013 Accepted 17 November 2013 Available online 26 November 2013 Keywords: Gold nanoparticles Protein–DNA interactions EMSA Fluorescent anisotropic Forkhead box protein

a b s t r a c t Forkhead box protein 1 (FoxA1) is a member of the forkhead family of winged helix transcription factors that plays pivotal roles in the development and differentiation of multiple organs and in the regulation of estrogen-stimulated genes. Conventional analytical methods—electrophoretic mobility shift assay (EMSA) and fluorescence anisotropy (FA)—as well as a gold nanoparticles (AuNPs)-based assay were used to study DNA binding properties of FoxA1 and ligand interruption of FoxA1–DNA binding. In the AuNPs assay, the distinct ability of protein–DNA complex to protect AuNPs against salt-induced aggregation was exploited to screen sequence selectivity and determine the binding affinity constant based on AuNPs color change and absorbance spectrum shift. Both conventional EMSA and FA and the AuNPs assay suggested that FoxA1 binds to DNA in a core sequence-dependent manner and the flanking sequence also played a role to influence the affinity. The EMSA and AuNPs were found to be more sensitive than FA in differentiation of sequence-dependent affinity. With the addition of a spin filtration step, AuNPs assay has been extended for studying small molecular ligand inhibition of FoxA1–DNA interactions enabling drug screening. The results correlate very well with those obtained using FA. Ó 2014 Published by Elsevier Inc.

Forkhead box protein 1 (FoxA1),1 also known as hepatocyte nuclear factor 3-alpha (HNF-3), is a member of the forkhead family of winged helix transcription factors that plays pivotal roles in the development and differentiation of multiple organs, including liver, pancreas, gut, breast, and prostate [1,2]. In addition to its roles in normal physiology, FoxA1 is important in diseases such as cancers. For example, FoxA1 has been found to be overexpressed in cancers such as breast and prostate [3,4], and its expression level is correlated with poor prognosis in prostate cancer patients [5,6]. FoxA1,

⇑ Corresponding authors. Address: Cancer Biology and Pharmacology, Genome Institute of Singapore, Agency for Science, Technology, and Research (A⁄STAR), Singapore. Fax: +65 6808 8305 (E. Cheung). Institute of Materials Research and Engineering, Agency for Science, Technology, and Research (A⁄STAR), Singapore. Fax: +65 6872 0785 (X. Su). E-mail addresses: [email protected] (E. Cheung), [email protected] (X. Su). 1 Abbreviations used: FoxA1, forkhead box protein A1; ERa, estrogen receptor a; EMSA, electrophoretic mobility shift assay; FA, fluorescence anisotropy; AuNPs, gold nanoparticles; ERE, estrogen receptor response element; NCI, National Cancer Institute; UV, ultraviolet; MWCO, molecular weight cutoff; dsDNA, double-stranded DNA. 0003-2697/$ - see front matter Ó 2014 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.ab.2013.11.017

as a sequence-specific transcription factor, interacts with chromatin as a monomer via the winged helix domain within its DNA binding region [7,8] and recognizes forkhead motifs with a core sequence of (G/A)(T/C)AAA(C/T)A [9,10]. Interestingly, FoxA1 was coined as a ‘‘pioneer’’ transcription factor when it was shown binding to the enhancer region of the liver-specific albumin gene before other transcription factors during liver cell development [3,11]. The pioneering activity of FoxA1 has subsequently been observed in other systems, including hormone-regulated transcription in cancer cells [12,13]. With recent advances in genomic technologies, such as ChIP– chip and ChIP–Seq, the genome-wide binding sites of FoxA1 have been mapped extensively in numerous systems and under different cellular conditions [14–17]. For example, tens of thousands of FoxA1 binding sites have been identified in breast cancer cells [18]. More important, a large fraction of these FoxA1 binding sites was found located in close proximity to estrogen receptor a (ERa) binding sites, suggesting that these two transcription factors may work together to regulate ERa-dependent transcription [3,19]. Indeed, studies by our group and others showed that FoxA1 is a key transcription factor in the estrogen signaling pathway that

96

FoxA1–DNA interaction and ligand inhibition study / K.M.M. Aung et al. / Anal. Biochem. 448 (2014) 95–104

functions as a pioneer factor in determining the binding, chromatin looping, and gene transcription mediated by ERa [14,18]. A detailed characterization of the DNA binding properties of FoxA1, therefore, is of great importance for us to understand how this factor regulates the transcriptional activity of ERa and future drug development for diseases such as breast cancer. Electrophoretic mobility shift assay (EMSA) and fluorescence anisotropy (FA) are conventional methods for studying protein– DNA interactions, both rely on the use of labeled DNA. EMSA requires on-gel separation of bound and unbound protein–DNA species [20]. To visualize the band shift, the DNA is usually labeled with biotin, radioactive isotopes, or fluorescent compounds. Although radioactive labeling is usually referred to as the gold standard, it has the drawbacks of safety, cost, and lifetime concerns. The non-radioactive labeling also suffers from relatively high cost [21]. FA, on the other hand offers a relatively quick and direct quantitation of protein–DNA binding without a separation step [22]. Once proteins are bound to labeled DNA, a larger complex will contribute to less tumbling movement and causes a lesser extent of light depolarization and, in turn, a high anisotropy value [23,24]. This method, however, has relatively low sensitivity (see Results and Discussion below) and requires a higher concentration of labeled DNA [22]. Sometimes additional tags of large size are required to amplify the signal [25,26]. Surface plasmon resonance spectroscopy [27–32] and other similar surface-sensitive analytical tools, such as quartz crystal microbalance [27,30] and dual polarization interferometry [33], offer label-free detection of protein–DNA interactions. However, the involvement of expensive equipment and skillful personnel for surface immobilization (an essential step for solid–liquid interface analysis) largely hinders their implementation for rapid screening and high-throughput analysis. In view of the importance in determining transcription factor– DNA interactions, a great deal of effort has been made to develop methodologies to complement the conventional methods (EMSA and FA) and the expensive equipment-based methods. To this end, our group has recently developed a series of gold nanoparticles (AuNPs)-based colorimetric assays for detecting protein– DNA interactions, exemplified by ERa and ERb and their response elements (EREs) [34–37]. Furthermore, these assays can be operated using a simple plate reader in a 96-well plate that allows these assays to be easily adopted in most laboratories. In many of these assays, one particular design exploited citrate ion-coated AuNPs as the sensing probe [35]. The stability of citrate ion-coated AuNPs is found to be largely associated with the formation of ER– ERE complexes. We have reasoned that the unique size and charge properties of the protein–DNA complexes provide efficient electrosteric protection to the AuNPs against salt-induced aggregation, which is detectable through particle solution color change and/or the spectrum shift of the localized surface plasmon resonances (LSPRs) peak. In this study, we extended the application of this complex sizeand charge-based homogeneous-phase AuNPs assay for interrogating FoxA1–DNA binding reactions to further validate the assay principle with a transcription factor beyond ERs. In addition, the assay protocol was modified for quantitative determination of the FoxA1–DNA binding affinity constant (Kd), which has not been reported. EMSA and FA were also performed in parallel to compare the performance of the assays. More important, in this study we readily extended the AuNPs assay for studying ligand inhibition of protein–DNA interactions that is important for drug discovery. With a modification of the assay protocol (i.e., the addition of spin filtration), the assay is capable of fast screening of ligand inhibition in an instrumentation- and label-free manner. With the parallel study using three methods, we had a chance to compare the conventional methods and the AuNPs assay, and from the results we

conclude that the AuNPs assay is an appealing technique that can facilitate gene transcription study and the drug discovery process. Materials and methods Purification of HisMBP FoxA1 protein Full-length FoxA1 complementary DNA (cDNA) was cloned into a pHISMBP (Addgene) expression vector via the Gateway cloning system (Invitrogen) as described by the manufacturer. FoxA1 was expressed as a HisMBP fusion protein in BL21(DE3) cells at 18 °C for 18 h of 0.5 mM isopropyl b-D-thiogalactopyranoside (IPTG) induction in TB (Terrific Broth) medium. Cells were collected by centrifugation, resuspended in a lysis buffer containing 50 mM Tris [pH 8.0], 300 mM NaCl, and 30 mM imidazole, and then sonicated on ice. Fusion proteins were initially purified from cell lysates using a nickel column equilibrated with the lysis buffer and eluted with the same buffer supplemented with 300 mM imidazole. A second purification step using ion exchange chromatography was performed by passing the sample through a Resource Q anion exchanger (GE Healthcare) and eluted in buffer containing 10 mM Tris–HCl and 1 M NaCl (pH 8.0) with a linear gradient from 0.1 to 1.0 M NaCl. Eluted fractions were collected, pooled, dialyzed against storage buffer (10 mM Tris–HCl, 100 mM NaCl, and 2 mM tris(2-carboxyethyl)phosphine [TCEP], pH 8.0), and concentrated to approximately 100 lM using a Vivaspin 20 concentrator before storing at 80 °C until use. Mass spectrometry (see Fig. S1 in Online supplementary material) and Western blot analysis were performed to confirm the proper expression of the protein (data not shown). Oligonucleotides FoxA1 DNA probes (see Table 1 for sense strand sequences) were synthesized by Sigma–Proligo (Singapore). They were labeled with Cy5 and FAM for the EMSA and FA experiments, respectively, or were non-labeled for the AuNPs experiment. Prior to use, the complementary strands of DNA were annealed in 20 mM Tris buffer solution (pH 8.0, 50 mM KCl, and 50 mM MgCl2) to form double-stranded DNA. Small molecule ligands Five low molecular weight ligands (200–3000 Da) were obtained from the diversity, mechanistic, and natural products libraries procured from the National Cancer Institute (NCI, http:// dtp.nci.nih.gov/index.html). They are sulfonated azo dye, metal– oxide cluster, semicarbozone, and polycyclic compounds by nature (see Table 2 for full names and molecular weights). All ligands were prepared in dimethyl sulfoxide (DMSO). Gold nanoparticles synthesis HAuCl43H2O (99.99%) and trisodium citrate dihydrate (99.9%) were obtained from Aldrich for the AuNPs synthesis according to our previously published protocol [35]. All chemicals and materials

Table 1 FoxA1 probe sequences.

a

Probe

Probe sequencea

Probe 1

50 -GTACTGTAAATAAAACT-30

Probe 2

50 -TGCCAAGTAAATAGTGCAG-30

Negative probe

50 -CACTTTGTTTGCAAAGC-30

FoxA1 consensus sequences are underlined.

FoxA1–DNA interaction and ligand inhibition study / K.M.M. Aung et al. / Anal. Biochem. 448 (2014) 95–104

were used as received without further purification. Ultrapure water (18 MO, prepared from a Millipore Elix 3 purification system) was used as the solvent unless indicated otherwise. The average diameter of AuNPs was 13 nm and in a concentration range of 8 to 12 nM (batch-to-batch variation), calculated according to Beer’s law using the extinction coefficient of 2.7  108 M1 cm1 for 13 nm AuNPs. Electrophoretic mobility shift assay This assay was carried out to determine the equilibrium dissociation constants of FoxA1 with different wild-type probes 1 and 2. Binding reactions included 1 nM Cy5-labeled DNA and serially diluted concentrations of protein and incubated for 1 h at 4 °C in buffer containing 10 mM Tris–HCl (pH 8.0), 0.1 mg/ml bovine serum albumin, 50 lM ZnCl2, 100 mM KCl, 10% glycerol, 0.1% NP-40, and 2 mM b-mercaptoethanol. Reactions were run on 4% native gels with 1 Tris–glycine buffer (pH 8.3) at 4 °C under 200 V constant voltage for 15 min. The gels were scanned using a Typhoon 9140 PhosphorImager (GE Healthcare). Band intensities of bound and unbound samples were quantified using ImageQuant TL software (GE Healthcare) and used to calculate the fraction bound of each probe based on three independent EMSA experiments. The equilibrium dissociation constants were then calculated by fitting the plot of DNA fraction bound versus protein concentration to the following equation, assuming 100% protein activity (Eq. 1) [38]:

f ¼

ðTF þ DL þ K d Þ 

qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ðTF þ DL þ K d Þ2  4DL  TF 2DL

97

(Tecan Trading, Switzerland) was used to measure the UV–visible absorption spectra of the AuNPs samples from 400 to 900 nm. Zeta potential measurements were performed on citrate ions-capped AuNPs in water, buffer (containing 20 mM KCl) with and without FoxA1, and FoxA1–DNA complex using a ZETA PLUS zeta potential analyzer (Brookhaven Instruments, USA). For Kd measurement, titration experiments were performed. Specifically, 500 nM FoxA1 was incubated with DNA probes of increasing concentrations at 0, 50, 100, 200, 300, 400, 500, 600, 700, 800, and 1000 nM. The incubation mixtures were then mixed with AuNPs similar as above. On mixing, UV–visible spectra were recorded and the relative absorption at 700 nm (A700) was plotted against DNA concentration. The resulting curve was fitted using a Langmuir isotherm to obtain the Kd. To detect ligand inhibition, FoxA1 was first incubated with 8fold excess of ligands for 40 min, followed by incubation with DNA probe for 20 min. Prior to the addition to AuNPs, the incubation mixtures were subjected to filtration with Vivaspin 500 (5000 MWCO [molecular weight cutoff]) at 14,000 rpm for 10 min to remove the excess or unreacted ligands. The retained solution (containing proteins and/or protein–DNA complex) was then incubated with AuNPs for colorimetric testing.

Results and discussion Determining HisMBP–FoxA1 affinity and specificity to DNA probe using EMSA

;

ð1Þ

where f represents the fraction of DNA bound, TF and DL refer to the concentration of protein and DNA used, respectively, and Kd is the dissociation constant. This is a modified Langmuir isotherm for DNA concentration substantially smaller than the protein. Fluorescence anisotropy To determine the binding activity of HisMBP–FoxA1 to various FoxA1 probes containing its cognate DNA element, FA assay was used. The sequences of the two wild-type probes and a mutant FAM-labeled probe used were as described earlier. The assays were carried out in 384-well microplates (Corning) in which varying concentrations of protein (0–2500 nM) were incubated with 10 nM labeled probe in PBS buffer (0.01 M phosphate buffer, 2.7 mM KCl, and 137 mM NaCl, pH 7.4) for 20 min at room temperature. Fluorescence anisotropy was measured using a Synergy 2 Multi-Mode Microplate Reader with a 485/20 excitation filter and a 528/20 emission filter. The equilibrium dissociation constants for each wild-type probe were subsequently calculated by fitting the FoxA1 concentration versus the FA plot using a Langmuir isotherm in Origin Pro 8 software. Standard error values (precision of the fitted values) are also obtained from the software after fitting. Gold nanoparticles assay Ultraviolet (UV)-transparent microplates (96 wells, clear, flat bottom; BD Falcon, USA) were used as reaction carriers. FoxA1 and DNA probes were mixed at a molar ratio of 1:1 at room temperature for 20 min in 10 mM Tris–HCl buffer solution (pH 7.4) containing 80 mM KCl, 0.15 mM ethylenediaminetetraacetic acid (EDTA), 0.3 mM dithiothreitol (DTT), and 1% glycerol. Then 25 ll of the complex solutions was mixed with 75 ll of the as-synthesized AuNPs to make up a final KCl concentration of 20 mM. The final concentrations of FoxA1 and DNA in the AuNPs solution mixture were 125 nM each. A Tecan Infinite M200 plate reader

To test the DNA binding activity of purified recombinant fulllength FoxA1 protein, two DNA probes containing the FoxA1 core consensus binding sequence (G/A)(T/C)(A/C)AA(C/T)A or mutant sequence were used [7,39] (Table 1). The 17-bp probe 1 sequence was obtained from one of the binding sites derived from our previous ChIP–Seq of FoxA1 in breast cancer cells that was associated with the ZBTB16 gene [40]. The 19-bp wild-type (probe 2) sequence was derived from the FoxA1 binding site located in the proximal promoter region of the lung-specific gene, Scgb1a1 [41–43]. The negative probe, designed as a negative control probe to test the selectivity of FoxA1–DNA binding, carries all mutated base pairs from probe 1 and 2. EMSA was first performed to visualize the binding of the FoxA1 protein to the fluorescence-labeled (Cy5) DNA probes 1 and 2. As shown in Fig. 1, there is an increase in mobility shift of the DNA with increasing protein concentrations, an indication of protein–DNA complex formation. Notably, smeary bands were observed instead of single bands, suggesting that several FoxA1–DNA complexes of differing stoichiometry were present in the reaction, or maybe that the protein contained more than one binding species (i.e., heterogeneous protein sample) [20] and, thus, multiple protein–DNA complexes from different protein species are formed. Because the HisMBP–FoxA1 protein sample had previously been shown to contain a mixture of major 70-kDa and minor 90-kDa proteins (see Fig. S1 in Supplementary material), it could account for the smeary band observed in Fig. 1. In addition, previous literature has reported that FoxA1 binds DNA at a stoichiometry of 1:1 [9]. This further affirms our hypothesis that the smeary bands are due to FoxA1–DNA complexes of different protein species instead of complexes of different stoichiometric ratio. To quantify the binding affinity of FoxA1 protein to different DNA probes, the band intensity of each bound and unbound sample was quantified and calculated as the fraction of DNA bound. This allowed for the dissociation constants (Kd) of each probe to be determined, based on the plot of DNA fraction bound versus protein concentration, as shown on the right of Fig. 1. Our results showed that probe 1 exhibited a higher affinity (47.82 ± 5.45 nM)

98

FoxA1–DNA interaction and ligand inhibition study / K.M.M. Aung et al. / Anal. Biochem. 448 (2014) 95–104

Fig.1. Determination of FoxA1 affinity for DNA probes via gel shift assay. A, B EMSA experiment showing the binding activity of varying concentrations of FoxA1 fusion protein with 1 nM Cy5 fluorescence-labeled probe 1 (A) and probe 2 (B). On the left are representative EMSA gel images showing increased intensity of the mobility shift with increasing protein concentrations. On the right are the averaged graphs used to determine the dissociation constant of each probe using data quantified from the respective gels.

than probe 2 (241.66 ± 55.24 nM), a difference of approximately 5fold (Table 3). Because the protein contains two components of 70 and 90 kDa, we refer to these affinity values (and others obtained by FA and AuNPs in subsequent sessions) as apparent affinity. These apparent affinity values would reflect the binding strength of the 70-kDa protein better because this protein is of the dominant amount, as shown in Fig. S1D. Determining HisMBP–FoxA1 affinity and specificity to DNA probe using FA Next, FA assay was also performed to complement the FoxA1 probe affinity study by EMSA. As mentioned in the introductory paragraphs, FA offers a relatively quick and direct quantitation of protein–DNA binding activity. Unlike EMSA, no gel separation step of bound and unbound species is needed [22,23]. Once proteins are

Table 2 Ligands tested in AuNPs assay for detecting ligand inhibition of FoxA1–probe 1 binding.

L1 L2 L3 N1 N2

Name

NSC

Molecular weight (Da)

Quinobene Dawson Lomofungin Picolinaldehyde 2-Bromo-1H-phenalen-1-one

638352 622124 106995 95678 177866

935 3015 314 196 259

bound to the free labeled DNA, a larger complex will form. This contributes to less tumbling movement of the complex and causes a lesser extent of light depolarization and, in turn, a high anisotropy value. A higher anisotropy, therefore, would imply a higher proportion of DNA bound. As shown in Fig. 2A, when FAM-labeled

99

FoxA1–DNA interaction and ligand inhibition study / K.M.M. Aung et al. / Anal. Biochem. 448 (2014) 95–104

sequence of the wild-type probe, was incubated with FoxA1. As shown in Fig. 2A, very little binding was observed for the negative probe compared with probes 1 and 2. This indicates that the core sequence is essential to form strong complexes with FoxA1 proteins. For the negative probe, protein binding was detected only when the protein concentration was higher than 100 nM, presumably due to sequence independence.

Table 3 Kd (nM) values obtained by different methods. Method

Probe 1

Probe 2

EMSA FA AuNPs

47.82 ± 5.45 33.48 ± 10.76 61.90 ± 17.40

241.66 ± 5.24 84.67 ± 26.59 341.10 ± 8.70

probes 1 and 2 were incubated with increasing concentrations of FoxA1 protein, the anisotropy increased at varying levels, suggesting the difference in the protein affinity for different probes. To quantitate the Kd for probes 1 and 2, two independent FA assays were performed. Consistently, probe 1 (Kd = 33.5 ± 10.8 nM) showed a better affinity than probe 2 (Kd = 84.7 ± 26.6 nM); however, the difference this time is approximately 2.5 times. Taken together, both EMSA and FA results show that despite probes 1 and 2 sharing identical core sequence, they display differences in FoxA1 binding affinity. This suggests that the flanking sequence adjacent to the main binding site may have a role in influencing the degree of FoxA1 binding. Indeed, previous studies have shown that besides the DNA binding domain of FoxA1 binding to DNA, the wing regions of FoxA1 can also interact with DNA at the minor groove for higher affinity and stability [9,10]. Hence, the apparent difference in the flanking sequence (recognized by the wing) could possibly account for the slight difference in affinity observed between the two probes. To test FoxA1 binding specificity to DNA probe, the mutant probe (i.e., negative probe), with all base pair mutations at the core

We previously reported the principle and procedure of the AuNPs assay for protein–DNA complex formation [35]. In this article, we now extend the principle to detect not only protein–DNA complex formation but also ligand inhibition (Fig. 3). Basically, this assay exploits the phenomenon that protein–DNA complexes can coat onto negatively charged AuNPs to provide protective forces against salt (e.g., KCl)-induced aggregation, by electrosteric protection, due to the large size and concentrated negative charges of protein–DNA complexes. To develop an AuNPs assay protocol for HisMBP–FoxA1, we first determined a suitable protein concentration (Fig. 4A). AuNPs in water are stable and highly dispersed, as shown by a sharp surface plasmon peak at 520 nm. When AuNPs are exposed to a buffer solution containing 80 mM KCl, the particles are largely aggregated, as indicated by the decrease in absorbance at 520 nm and the appearance of absorption at a longer wavelength (700 nm). The addition of HisMBP–FoxA1 retarded AuNPs aggregation (presumably due to the coating of the protein

B 0.16

0.14

0.14

0.12 0.10 0.08 0.06

C 0.16 0.14 Fluorescence anisotropy

0.16

Fluorescence anisotropy

Fluorescence anisotropy

A

Detection of HisMBP–FoxA1–DNA complex formation using AuNPs colorimetric assay

0.12 0.10 0.08 0.06

0.12 0.10 0.08 0.06

0.04 0

25

50 75 100 125 250 500 750 1000 1500 2000 2500

FoxA1 concentration (nM)

0.04

0

500

1000

1500

2000

FoxA1 concentration (nM)

2500

0.04

0

500

1000

1500

2000

2500

FoxA1 concentration (nM)

Fig.2. Comparison of FoxA1 affinity for DNA probes via FA assay. (A) Binding profile of FoxA1–DNA with 10 nM FAM fluorescence-labeled probe 1, probe 2, and negative probe. Increasing concentrations of FoxA1 increase the FA, indicating FoxA1–DNA complex formation. Data represent the averages of five measurement readings. Kd plots that were used to determine the dissociation constants of probe 1 (B) and probe 2 (C) are shown using the protein–DNA binding data obtained from panel A.

Probe 1: 5’-GTACTGTAAATAAAACT-3 Probe 2: 5’-TGCCAAGTAAATAGTGCAG-3’ Negative Probe: 5’-TGCCAAGAGGGTAGTGCAG-3’

Fig.3. Schematic diagram of AuNPs assay principle without (route A) and with (route B) ligand loading.

100

FoxA1–DNA interaction and ligand inhibition study / K.M.M. Aung et al. / Anal. Biochem. 448 (2014) 95–104

A

0.6

In buffer soluon

Absorbance

0.5 0.4 0.3 0.2 0.1

[FoxA1]/nM 0 100 200 400 500 600 800

in water

0 400

500

600

700

800

900

800

900

Wavelength (nm)

B

0.6

In buffer soluon

Absorbance

0.5 0.4 0.3

a

0.2

b c d

in water

0.1 0 400

500

600

700

Wavelength (nm)

C

water

buffer soluon (80 mM KCl)

Zeta potenal (mV)

in FoxA1probe1 complex

in FoxA1

no biosamples

0 -5 -10 -15 -20 -25 -30 -35 -40

Fig.4. AuNPs assay to screen FoxA1–DNA complex formation. (A) UV–visible spectra of AuNPs in the presence of FoxA1 of increasing concentrations in binding buffer (10 mM Tris–HCl) containing 80 mM KCl. (B) UV–visible spectra of AuNPs mixed with FoxA1–probe 1 (a), FoxA1–probe 2 (b), FoxA1–negative probe (c), and FoxA1 in buffer solution (d). The final KCl in the buffer solution is 20 mM. The FoxA1 concentration and DNA probe concentration in panel B are both 500 nM. In both panels A and B, AuNPs in water and in buffer without biological samples are shown as reference. (C) Zeta potential measurement of different AuNPs samples in water and buffer solution.

to the particles) in a protein concentration-dependent manner. The higher the protein concentration, the more stable the particles became. Ultimately, 500 nM FoxA1 was chosen for detecting the protein–DNA complex formation because the AuNPs undergo intermediate aggregation that offers the best possibility to detect protein–DNA complex formation through increased stabilization. To detect the FoxA1–DNA complex formation, the protein and DNA were incubated at a 1:1 ratio (500:500 nM). The complex incubation solution was then added into AuNPs. Fig. 4B shows the UV–visible adsorption spectra of AuNPs exposed to FoxA1 (500 nM), FoxA1–probe 1, FoxA1–probe 2, and FoxA1–negative probe (1:1 molar ratio) in incubation solutions compared with those of AuNPs (in water) and in 80 mM KCl-containing solution

as references. It is very obvious that in the presence of DNA probes, the AuNPs become more stable (more dispersed) relative to the FoxA1 alone, signifying the formation of protein–DNA complex. The degree of stabilization appears to be sequence dependent, that is, probe 1 > probe 2 > negative control DNA probe. Thus, it can be inferred that probe 1 has a higher apparent affinity than probe 2, as demonstrated by the EMSA and FA assay. Similar to the FA result, a certain degree of binding was also detected for the negative control DNA probe, presumably due to the sequence-independent contact. But unlike the FA, where ultra-weak nonspecific FoxA1–DNA interactions can be observed at a higher relative protein concentration of at least 10 times more than the DNA (100 nM protein:10 nM DNA), this AuNPs assay can detect sequence-independent protein–DNA binding at a lower relative protein concentration of 1 mol protein:1 mol DNA (500 nM protein:500 nM DNA). To confirm that the electrosteric effect exerted by protein–DNA complex is responsible for the differential AuNPs aggregation profile, zeta potential was measured to determine the surface charge of AuNPs under different sample treatment (Fig. 4C). Because FoxA1 is slightly positively charged at tested pH 8.0 (isoelectric point = 8.93) [44], the AuNPs coated with FoxA1 exhibit a much lower negative charge density (14.5 mV) than bare citrate ioncoated AuNPs (35.0 ± 2.3 mV) due to the charge neutralization and/or displacement of the citrate ions. When FoxA1–DNA complexes are formed and coat on the AuNPs, the particles gain more negative charges from the double-stranded DNA (dsDNA) in the FoxA1–DNA complex (30.6 ± 1.6 mV) and, thus, result in enhanced particle stability. Determining HisMBP–FoxA1 affinity to DNA using AuNPs colorimetric assay Because the degree of particles being protected is highly associated with the amount of protein–DNA complex, we next performed titration experiments to determine the binding affinity constant, Kd. Unlike the EMSA and FA assay (Figs. 1 and 2), where a fixed amount of labeled DNA was titrated with increasing FoxA1 concentrations, here we instead fixed FoxA1 concentration but increased DNA probe concentration. This arrangement was adopted because dsDNA has no affinity to AuNPs [45] and, thus, will not affect AuNPs aggregation at a wide range of tested concentrations. Fig. 5A shows a UV–visible spectrum of AuNPs at a fixed FoxA1 concentration of 500 nM but with increasing DNA probe 1. The relative absorbance intensity at 700 nm that is related to the amount of complex formed was plotted as a function of DNA concentration and fitted using a Langmuir isotherm (Fig. 5B). Relative absorbance values were obtained by subtracting the absorbance value at 0 nM DNA concentration from A700. The affinity constant for probe 1 is 61.9 ± 17.4 nM. In this calculation, 700 nm was chosen because it is the peak of aggregated particles that could be the most representative, inversely, of the amount of FoxA1–DNA complex formed. We also used relative absorbance at wavelengths of 650, 750, 800, and 850 nm to calculate the Kd values (see Fig. S2 and Table S1 in Supplementary material). A comparable Kd value was obtained using 750 nm data. At other wavelengths (i.e., 650, 800, and 850 nm), the Kd values are less reliable because the absorbances at those wavelengths are less well represented of aggregated particles and, thus, are less reflective of the amount of FoxA1–DNA complex formed. A similar experiment was performed for probe 2, which resulted in a Kd of 341.1 ± 68.7 nM using relative A700 (Fig. S3) that is 5.5 times larger than that for probe 1. These affinity values are in general agreement with the EMSA and FA results that probe 1 has a higher affinity than probe 2 by approximately 5-fold. It should be noted that the absolute Kd values obtained by the different methods varied from each other. This is because different

FoxA1–DNA interaction and ligand inhibition study / K.M.M. Aung et al. / Anal. Biochem. 448 (2014) 95–104

A

0.6

in buffer

Absorbance (a.u.)

0.5

FoxA1 (500 nM):probe 1 (x nM) x (nM) 0 50 200 300 400 500 600 700 800 1000

0.4 0.3 0.2

AuNPs in water

0.1

500

600

700

800

900

Wavelength (nm)

B

Relative A700

0.20 0.15 0.10 0.05 0.00 0

200

400

600

800

on the overall size (and charge for the latter method) of the complex mixtures, whereas EMSA allows ‘‘visualizing’’ different complex species of different size and mobility. Determining ligand inhibition of FoxA1–DNA interaction using AuNPs colorimetric assay

0 400

101

1000

Probe 1 concentration (nM) Fig.5. AuNPs titration experiments for Kd measurement for probe 1. (A) UV–visible spectra of AuNPs in the presence of FoxA1–probe 1 incubation mixture, where FoxA1 is fixed at 500 nM and DNA (probe 1) concentration is increasing from 0 to 1000 nM. The final KCl in the buffer solution (10 mM Tris–HCl) is 20 mM. (B) Langmuir fit of the titration curve, that is, the amount of complex formed (determined as relative absorbance at 700 nm) versus DNA conentration.

assays have different experimental principles and, thus, the Kd values obtained can be considered as partition coefficients or as ‘‘apparent’’ under the conditions of a particular assay but not considered as experimentally measured Kd values as ‘‘absolute’’ [46,47]. In the current study, the mathematical model used for calculating Kd for EMSA was a modified Langmuir isotherm, which approximates to the Langmuir model when the DNA concentration is small compared with protein. The Kd values of FA and AuNPs assays were calculated using a Langmuir model. Based on the parallel measurement under similar binding conditions (Tris–HCl buffer [pH 8.0] containing 100 mM KCl) and using approximate fitting models, we compared the performance of each detection method and made the following observations. First, EMSA and AuNPs assay have similar sensitivity to discriminate binding affinity (5.0–5.5 times of difference for probes 1 and 2 measured by EMSA and AuNPs), whereas FA assay is less powerful in differentiating the affinity difference (only 2.5 times of difference measured by FA). Second, AuNPs are more sensitive in detecting sequence-independent or nonspecific protein–DNA contact (at a protein:DNA ratio of 1:1) than FA (at a ratio > 10:1) because dsDNA itself has negligible affinity to AuNPs, but protein–DNA complex can easily coat on AuNPs. In the FA assay that measures size difference between labeled DNA and protein–DNA complex, at lower protein concentrations, no substantial complex can form to increase the size of the labeled DNA. As for EMSA, it was not able to detect sequence-independent binding, presumably due to the on-gel separation step that can dissociate weak transient binding. Third, FA and AuNPs assays measure protein–DNA complex based

The identification and characterization of small molecules that inhibit protein–DNA interactions can potentially lead to the discovery of new therapeutic drugs. Using fluorescence anisotropic assay, we recently screened and identified a number of high-affinity ligands from the NCI Mechanistic Set that interrupt FoxA1–DNA binding (data not shown). The binding affinities of these compounds to FoxA1 have been confirmed by the enhanced protein thermal stability and by Biacore T200 measurement (unpublished results in our laboratory). In this study, using three of the affinity compounds (L1, L2, and L3) and two non-inhibitor compounds (N1 and N2) as examples (Table 2), we tested the suitability of the AuNPs assay for fast screening ligand inhibition of protein– DNA (FoxA1–probe 1) interaction. According to the FA result, L1 and L2 are strong inhibitors that can totally diminish FoxA1–DNA binding, generating low fluorescence anisotropy value similar to that of using a non-labeled DNA to complete the FoxA1-labeled DNA complex formation. L3 is a weaker inhibitor (80% inhibition relative to that with a non-labeled DNA). N1 and N2 have a barely detectable inhibition effect. Fig. 6 (top panel) shows the AuNPs assay results using the exact protocol for FoxA1–probe 1 except that the FoxA1 was first loaded with 8 times excess ligands for 40 min prior to incubation with the DNA probe. In the presence of the three positive (or affinity) ligands, the AuNPs spectrum largely moved toward that of FoxA1 alone (signifying particle aggregation), perhaps indicative of the ligand interruption of FoxA1 to form FoxA1–DNA complex, detected as particles aggregation due to the lack of FoxA1–DNA complex protection. However, when the two negative ligands were tested, where the AuNPs should remain well dispersed similarly as they are in FoxA1–probe 1 complex, a certain degree of particle aggregation (spectrum moving toward that with FoxA1 alone) was also observed unexpectedly, especially for N1. Because these negative ligands are known to have a barely detectable inhibition effect, protein–DNA complex should form and remain available to protect the AuNPs, similarly as the complex in the absence of any ligands (the blue curve). To determine the reason for the unexpected aggregation, we examined the chemical structures of the compounds and realized that some of them contain amines and hydroxyl and thiol groups that would have certain affinity to gold through SH–Au interaction and (O–Au, N–Au) coordination chemistry. If these ligands are attached to the AuNPs, they will hinder the coating of the FoxA1– DNA complex to the particle. In a worse scenario, if the ligand itself can aggregate the particle due to the presence of multiple functional groups, it will further rupture the ground principle of this assay that is to rely on the coating of protein–DNA complex to AuNPs to provide protection forces. Thus, we examined the behavior of AuNPs with the tested ligands (L1–L3, N1, and N2) in water (Fig. 7A). Of the five tested ligands, we found that N1 can largely aggregate the particles in water, rendering immediate AuNPs color change from red to blue, associated with the significant spectrum shift. It is not surprising because this ligand contains both SH and NH2 groups that could crosslink the AuNPs. Although other ligands do not induce detectable aggregation, they could have coated on the particles, as shown by the intensity drop at the peak wavelength of 520 nm. In buffer solution that contains 80 mM KCl, the ligand effect is not clearly differentiated among all ligands because the AuNPs have been largely aggregated (peak wavelength moved to 660 nm) due to the strong salt screening of the surface

102

FoxA1–DNA interaction and ligand inhibition study / K.M.M. Aung et al. / Anal. Biochem. 448 (2014) 95–104 0.5 L1 w/o Spin

N2 w/o spin

N1 w/o spin

L3 w/o spin

L2 w/o spin

Absorbance

0.4 0.3 0.2 0.1 0 400

500

600

700

400

800

500

Wavelength (nm)

600

700

800

400

500

600

0.5

700

L2 with spin

L1 with spin

800

400

500

600

700

800

400

500

Wavelength (nm)

Wavelength (nm)

Wavelength (nm)

600 700 Wavelength (nM)

900

N2 with spin

N1 with spin

L3 with spin

800

Absorbance

0.4 0.3 0.2 0.1 0

400

500

600

700

800

400

500

Wavelength (nm)

600

700

800

400

500

600

700

Wavelength (nm)

Wavelength (nm)

800

400

500

600

700

Wavelength (nm)

800

400

500

600

700

800

900

Wavelength (nm)

Fig.6. AuNPs assay for detecting ligand inhibition of FoxA1–probe 1 binding. Top panels: No spin filtration. Bottom panels: Addition of spin filtration after FoxA1–ligand– probe 1 incubation prior to AuNPs test. Ligands 1 to 3 (L1–L3) are positive ligands that can interrupt the complex formation. N1 and N2 are negative liagnds. Each panel shows three curves (from left to right): AuNPs in FoxA1–probe 1 complex in 10 mM Tris–HCl buffer solution (80 mM KCl) (blue solid line); AuNPs in FoxA1–ligand–probe 1 complex in buffer solution (pink dashed line); AuNPs in FoxA1 solution only (green solid line). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

A

1.4 Without spin-filter

1.2

L1

Absorbance (a.u.)

1

L2

L3

N1

N2

N1’

With spin-filter

0.8

N1”

0.6 L1 L2

0.4 0.2 0

N1

L3 N2 400

500

600

700

800

Wavelength (nm)

B

0.5

In buffer

Absorbance

0.4

b

0.3

b’

a a’ 0.2

In water 0.1

0

400

500

600

700

800

900

Wavelength (nm)

Fig.7. AuNPs’ stability in ligands and spin filtration removal of ligands. (A) AuNPs spectrum in water and in the presence of 2.5 lM ligands (L1–L3, N1, and N2). The insets are the color photos of the respective AuNPs solutions. The color photo of N10 and N100 are the AuNPs solution where the N1 ligand was separated using spin filtration at 14,000 rpm for 10 min and 6000 rpm for 30 min, respectively. (B) AuNPs spectrum in the presence of FoxA1–probe 1 complex (a and a0 ) and FoxA1 alone (b and b0 ) without (solid lines) and with (dashed lines) spin filtration at 14,000 rpm for 10 min. The FoxA1 concentration is 600 nM. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

charge (see Fig. S4 in Supplementary material). However, a zoomin view of spectra at the peak wavelength also shows a slight intensity drop in the presence of various ligands that could signify the coating of the ligands to AuNPs. The potential intrinsic affinity of

ligands to AuNPs makes this bare AuNPs assay principle invalid for screening small ligand inhibition based on the existing protocol. To circumvent this problem, we introduced a spin filtration step after FoxA1–ligand–DNA incubation to remove unbound and/or excess ligands prior to AuNPs testing. We first established the spin filtration conditions (speed and time of separation) for efficient removal of the small molecular weight ligands and the effect to FoxA1 and FoxA1–DNA complex. Using the Vivaspin 500 (5000 MWCO) and spinning at 14,000 rpm for 10 min, or 6000 rpm for 30 min, all free ligands (at a tested concentration of 2.5 lM) can be efficiently removed from water, the reconstituted solution in the tube contains no detectable ligand when they are mixed with AuNPs, and the color of the AuNPs remains red (e.g., for N1 in Fig. 7A, see images N10 and N100 ). For FoxA1 protein, a certain degree of protein loss was observed after the spin filtration (Fig. 7B) that could be due to some protein sticking on the membrane. For the complex of FoxA1–probe 1, with and without this separation treatment, the complex remains stable and retrievable from the remaining solution in the tube, as evidenced by the retainable AuNPs stability (Fig. 7B). The filter membrane in the Vivaspin 500 is PES (polyethersulfone) membrane, which is known to cause protein loss due to hydrophobicity [48,49]. On the other hand, the protein–DNA complex could be more hydrophilic; thus, little loss to the membrane was observed. With the confirmation that spin filtration can remove unbound or excess ligands without affecting the preformed protein–DNA complex, this separation step was added into the protocol of testing ligand inhibition of protein–DNA interactions (Fig. 6, bottom panel). Much clearer opposite trends are now observed for all three positive ligands (inhibitors) versus the two negative ligands (noninhibitors). All three inhibitors are detected by their interruption of FoxA1–probe 1 formation, causing the shift of the AuNPs spectrum toward that with FoxA1 alone. The degree of the shift is smaller for L3, which is known to be a weaker inhibitor. All negative ligands gave no detectable effects, as shown by the nearly overlapped AuNPs spectrum in the presence of protein–DNA complex. With the improved protocol with the addition of spin filtration, we can unambiguously screen inhibitors and non-inhibitors by comparing the AuNPs spectrum in the presence of FoxA1–DNA–ligand versus FoxA1 alone.

FoxA1–DNA interaction and ligand inhibition study / K.M.M. Aung et al. / Anal. Biochem. 448 (2014) 95–104

Conclusion EMSA and FA are traditional methods for studying protein–DNA interactions. The AuNPs aggregation assay is a complementary method that allows quick screening of protein–DNA binding due to its label-free, fast, and easy-to-use features. With EMSA, fractional protein–DNA complexes of different binding ratios or different complex species can be ‘‘visualized’’ based on the mobility of complex bands. AuNPs assay and FA can quantify binding stoichiometry. For FoxA1, all three methods confirm that flanking sequence of its DNA probes plays a role to control the protein binding affinity. EMSA and AuNPs assay have similar sensitivity in discriminating sequence-dependent binding affinity, whereas FA assay is relatively less powerful to discriminate affinity difference. Coupled with a spin filtration step, the AuNPs assay became valid for screening ligand inhibition to protein–DNA interactions that is of practical significance for drug discovery research. Acknowledgment X.S. and E.C. acknowledge the Agency for Science, Technology, and Research (A*STAR), Singapore, for financial support (JCO 1131CFG001). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ab.2013.11.017. References [1] S. Belikov, P.H. Holmqvist, C. Astrand, O. Wrange, FoxA1 and glucocorticoid receptor crosstalk via histone H4K16 acetylation at a hormone regulated enhancer, Exp. Cell Res. 318 (2012) 61–74. [2] J.R. Friedman, K.H. Kaestner, The FoxA family of transcription factors in development and metabolism, Cell. Mol. Life Sci. 63 (2006) 2317–2328. [3] M.A. Augello, T.E. Hickey, K.E. Knudsen, FOXA1: master of steroid receptor function in cancer, EMBO J. 30 (2011) 3885–3894. [4] G.M. Bernardo, R.A. Keri, FOXA1: a transcription factor with parallel functions in development and cancer, Biosci. Rep. 32 (2012) 113–130. [5] J. Gerhardt, M. Montani, P. Wild, M. Beer, F. Huber, T. Hermanns, M. Muntener, G. Kristiansen, FOXA1 promotes tumor progression in prostate cancer and represents a novel hallmark of castration-resistant prostate cancer, Am. J. Pathol. 180 (2012) 848–861. [6] B. Sahu, M. Laakso, K. Ovaska, T. Mirtti, J. Lundin, A. Rannikko, A. Sankila, J.P. Turunen, M. Lundin, J. Konsti, T. Vesterinen, S. Nordling, O. Kallioniemi, S. Hautaniemi, O.A. Janne, Dual role of FoxA1 in androgen receptor binding to chromatin, androgen signalling, and prostate cancer, EMBO J. 30 (2011) 3962– 3976. [7] H. Nakshatri, S. Badve, FOXA1 in breast cancer, Exp. Rev. Mol Med. 11 (2009) e8. [8] X. Qian, R.H. Costa, Analysis of hepatocyte nuclear factor-3b protein domains required for transcriptional activation and nuclear targeting, Nucleic Acids Res. 23 (1995) 1184–1191. [9] L.A. Cirillo, K.S. Zaret, Specific interactions of the wing domains of FOXA1 transcription factor with DNA, J. Mol. Biol. 366 (2007) 720–724. [10] S.S. Myatt, E.W. Lam, The emerging roles of forkhead box (Fox) proteins in cancer, Nat. Rev. Cancer 7 (2007) 847–859. [11] K.S. Zaret, J.S. Carroll, Pioneer transcription factors: establishing competence for gene expression, Genes Dev. 25 (2011) 2227–2241. [12] L.A. Cirillo, C.E. McPherson, P. Bossard, K. Stevens, S. Cherian, E.Y. Shim, K.L. Clark, S.K. Burley, K.S. Zaret, Binding of the winged-helix transcription factor HNF3 to a linker histone site on the nucleosome, EMBO J. 17 (1998) 244–254. [13] L.A. Cirillo, F.R. Lin, I. Cuesta, D. Friedman, M. Jarnik, K.S. Zaret, Opening of compacted chromatin by early developmental transcription factors HNF3 (FoxA) and GATA-4, Mol. Cell 9 (2002) 279–289. [14] J.S. Carroll, X.S. Liu, A.S. Brodsky, W. Li, C.A. Meyer, A.J. Szary, J. Eeckhoute, W. Shao, E.V. Hestermann, T.R. Geistlinger, E.A. Fox, P.A. Silver, M. Brown, Chromosome-wide mapping of estrogen receptor binding reveals long-range regulation requiring the forkhead protein FoxA1, Cell 122 (2005) 33–43. [15] M. Lupien, J. Eeckhoute, C.A. Meyer, Q. Wang, Y. Zhang, W. Li, J.S. Carroll, X.S. Liu, M. Brown, FoxA1 translates epigenetic signatures into enhancer-driven lineage-specific transcription, Cell 132 (2008) 958–970. [16] Y. Zhang, T. Liu, C.A. Meyer, J. Eeckhoute, D.S. Johnson, B.E. Bernstein, C. Nusbaum, R.M. Myers, M. Brown, W. Li, X.S. Liu, Model-based analysis of ChIP– Seq (MACS), Genome Biol. 9 (2008) R137.

103

[17] E. Cheung, W.L. Kraus, Genomic analyses of hormone signaling and gene regulation, Annu. Rev. Physiol. 72 (2010) 191–218. [18] S.K. Tan, Z.H. Lin, C.W. Chang, V. Varang, K.R. Chng, Y.F. Pan, E.L. Yong, W.K. Sung, E. Cheung, AP-2c regulates oestrogen receptor-mediated long-range chromatin interaction and gene transcription, EMBO J. 30 (2011) 2569–2581. [19] C.S. Ross-Innes, R. Stark, A.E. Teschendorff, K.A. Holmes, H.R. Ali, M.J. Dunning, G.D. Brown, O. Gojis, I.O. Ellis, A.R. Green, S. Ali, S.F. Chin, C. Palmieri, C. Caldas, J.S. Carroll, Differential oestrogen receptor binding is associated with clinical outcome in breast cancer, Nature 481 (2012) 389–393. [20] L.M. Hellman, M.G. Fried, Electrophoretic mobility shift assay (EMSA) for detecting protein–nucleic acid interactions, Nat. Protoc. 2 (2007) 1849–1861. [21] N. Jullien, J.-P. Herman, LUEGO: a cost and time saving gel shift procedure, BioTechniques 51 (2011) 267–269. [22] B.J. Anderson, C. Larkin, K. Guja, J.F. Schildbach, Using fluorophore-labeled oligonucleotides to measure affinities of protein–DNA interactions, Methods Enzymol. 450 (2008) 253–272. [23] W.J. Checovich, R.E. Bolger, T. Burke, Fluorescence polarization—a new tool for cell and molecular biology, Nature 375 (1995) 254–256. [24] V.J. LiCata, A.J. Wowor, Applications of fluorescence anisotropy to the study of protein–DNA interactions, Methods Cell Biol. 84 (2008) 243–262. [25] Y. Qiao, H. Tang, G.R. Munske, P. Dutta, C.F. Ivory, W.J. Dong, Enhanced fluorescence anisotropy assay for human cardiac troponin I and T detection, J. Fluoresc. 21 (2011) 2101–2110. [26] X. Wang, M. Zou, H. Huang, Y. Ren, L. Li, X. Yang, N. Li, Gold nanoparticle enhanced fluorescence anisotropy for the assay of single nucleotide polymorphisms (SNPs) based on toehold-mediated strand-displacement reaction, Biosens. Bioelectron. 41 (2013) 569–575. [27] X.D. Su, C. Lin, S.J. O’Shea, H.F. Teh, W.Y.X. Peh, J.S. Thomsen, Combinational study of estrogen receptors–response elements interactions using SPR and QCM, Anal. Chem. 78 (2006) 5552–5558. [28] H.F. Teh, W.Y.X. Peh, X.D. Su, J.S. Thomsen, Characterization of protein–DNA interactions using SPR with various assay schemes, Biochemistry 46 (2007) 2127–2135. [29] N. Yang, X.D. Su, V. Tjong, W. Knoll, Evaluation of two- and three-dimensional streptavidin binding platforms for surface plasmon resonance spectroscopy studies of DNA hybridization and protein–DNA binding, Biosens. Bioelectron. 22 (2007) 2700–2706. [30] W.Y.X. Peh, E. Reimhult, H.F. The, X.D. Su, Understanding ligand binding effects on conformation of estrogen receptor a–DNA complex: a combinational QCMD and SPR study, Biophys. J. 92 (2007) 4415–4427. [31] X.D. Su, S.J. Neo, W.Y.X. Peh, J.S. Thomsen, Two-step antibody approach for SPR detection of protein–DNA interactions in nuclear extracts, Anal. Biochem. 376 (2008) 137–143. [32] S.J. Neo, X.D. Su, J.S. Thomsen, SPR study of cooperation interactions of estrogen receptor-a and transcription factor Sp1 to composite DNA, Anal. Chem. 81 (2009) 3344–3349. [33] H.Y. Song, W. Sun, S. Prabhakar, K.M.M. Aung, X.D. Su, Study sequence rules of estrogen receptor a–DNA interactions using dual polarization interferometry and computational modelling, Anal. Biochem. 433 (2012) 121–128. [34] Y.N. Tan, X.D. Su, J.Y. Lee, Sensing of transcription factor through controlledassembly of metal nanoparticles modified with segmented DNA elements, ACS Nano 4 (2010) 5101–5110. [35] Y.N. Tan, X.D. Su, E. Liu, J.S. Thomsen, A gold-nanoparticle-based assay for instantaneous detection of transcription factor–DNA interactions, Anal. Chem. 82 (2010) 2759–2766. [36] K.M.M. Aung, Y.N. Tan, X.D. Su, Interrogating estrogen receptor–DNA interactions using metallic nanoparticles and surface plasmon resonance technique, Aust. J. Chem. 64 (2011) 1288–1294. [37] Y.N. Tan, A. Lai, X.D. Su, Interrogating cooperative interactions of transcription factors with composite DNA elements using gold nanoparticles, Sci. Adv. Mater. (in press). [38] N. BabuRajendran, P. Palasingam, K. Narasimhan, W. Sun, S. Prabhakar, R. Jauch, P.R. Kolatkar, Structure of Smad1 MH1/DNA complex reveals distinctive rearrangements of BMP and TGF-b effectors, Nucleic Acids Res. 38 (2010) 3477–3488. [39] S. Belikov, C. Astrand, O. Wrange, FoxA1 binding directs chromatin structure and the functional response of a glucocorticoid receptor-regulated promoter, Mol. Cell. Biol. 29 (2009) 5413–5425. [40] P.Y. Tan, C.W. Chang, K.R. Chng, K.D. Wansa, W.K. Sung, E. Cheung, Integration of regulatory networks by NKX3-1 promotes androgen-dependent prostate cancer survival, Mol. Cell. Biol. 32 (2012) 399–414. [41] R.S. Peterson, D.E. Clevidence, H. Ye, R.H. Costa, Hepatocyte nuclear factor-3a promoter regulation involves recognition by cell-specific factors, thyroid transcription factor-1, and autoactivation, Cell Growth Differ. 8 (1997) 69–82. [42] Y. Guo, R. Costa, H. Ramsey, T. Starnes, G. Vance, K. Robertson, M. Kelley, R. Reinbold, H. Scholer, R. Hromas, The embryonic stem cell transcription factors Oct-4 and FoxD3 interact to regulate endodermal-specific promoter expression, Proc. Natl. Acad. Sci. USA 99 (2002) 3663–3667. [43] T. Kido, T. Tomita, M. Okamoto, Y. Cai, Y. Matsumoto, C. Vinson, Y. Maru, S. Kimura, FOXA1 plays a role in regulating secretoglobin 1a1 expression in the absence of CCAAT/enhancer binding protein activities in lung in vivo, Am. J. Physiol. Lung Cell. Mol. Physiol. 300 (2011) L441–L452. [44] Cell Signaling Technology, FOXA1 (human). .

104

FoxA1–DNA interaction and ligand inhibition study / K.M.M. Aung et al. / Anal. Biochem. 448 (2014) 95–104

[45] H. Li, L. Rothberg, Colorimetric detection of DNA sequences based on electrostatic interactions with unmodified gold nanoparticles, Proc. Natl. Acad. Sci. USA 101 (2004) 14036–14039. [46] US Environmental Protection Agency, Understanding Variation in Partition Coefficient, kd, values: Vol. 2: Review of Geochemistry and Available kd Values for Cadmium, Cesium, Chromium, Lead, Plutonium, Radon, Strontium, Thorium, Tritium (3H), and Uranium, Office of Radiation and Indoor Air, Washington, DC, 1999. [47] J. Leysen, X. Langlois, L. Heylen, A. Lammertsma, Receptors: binding assays, in: I. Stolerman, L. Price (Eds.), Encyclopedia of Psychopharmacology, SpringerVerlag, Berlin, 2010.

[48] B. Su, S. Sun, Z. Zhao, Polyethersulfone hollow fiber membranes for hemodialysis, in: Prof. Angelo Carpi (Ed.), Progress in Hemodialysis— from Emergent Biotechnology to Clinical Practice, 2011. ISBN: 978-953-307377-4, http://dx.doi.org/10.5772/22857, http://www.intechopen.com/books/ progress-in-hemodialysis-from-emergent-biotechnology-to-clinical-practice/ polyethersulfone-hollow-fiber-membranes-for-hemodialysis. [49] E. Celik, L. Liu, H. Choi, Protein fouling behavior of carbon nanotube/ polyethersulfone composite membranes during water filtration, Water Res. 45 (2011) 5287–5294.