Sensitive Detection of Transcription Factors Using Near-Infrared

Jan 29, 2014 - ... Near-Infrared Fluorescent Solid-Phase Rolling Circle Amplification ... of Bioelectronics, Southeast University, Nanjing 210096, Chi...
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Sensitive Detection of Transcription Factors Using Near-Infrared Fluorescent Solid-Phase Rolling Circle Amplification Junhuan Yin, Ping Gan, Fei Zhou, and Jinke Wang* State Key Laboratory of Bioelectronics, Southeast University, Nanjing 210096, China S Supporting Information *

ABSTRACT: This study describes a method for analyzing transcription factor (TF) activity, near-infrared fluorescent solid-phase rolling circle amplification (NIRF-sRCA). This method analyzes TF activity in four steps: (i) incubate DNA with protein sample and isolate TF-bound DNA, (ii) hybridize the TFbound DNA and rolling circle to DNA microarray, (iii) amplify the TF-bound DNA with sRCA that contains biotin-labeled dUTP, and (iv) detect sRCA products by binding of NIRF-labeled streptavidin and NIRF imaging. This method was validated by proof-of-concept detection of purified TF protein and cell nuclear extract. Detection of purified TF protein demonstrated that NIRFsRCA could quantitatively detect NF-κB p50 protein, and as little as 6.94 ng (∼140 fmol) of this protein was detected. Detection of nuclear extract revealed that NIRF-sRCA could specifically and quantitatively detect NF-κB p50 activity in HeLa cell nuclear extracts, and the activity of this TF in as little as 0.625 μg of nuclear extracts could be detected. Detection of nuclear extract also revealed that NIRF-sRCA could detect the relative activities of multiple TFs in HeLa cell nuclear extracts and the fold induction of multiple TFs in the TNFα-induced HeLa cell nuclear extracts. Therefore, this study provides a new tool for studying TFs.

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DNAs. Because of high sensitivity of these DNA amplification techniques, incomplete digestion resulted from low enzyme efficiency and nonspecific protein binding protection may easily result in false positives. On the other hand, the protein disassociation or displacement from DNA in enzyme digestion may result in false negatives. In addition, it is difficult to adapt these methods built in liquid reaction into the high throughput format for simultaneously detecting multiple TFs in a small amount of protein sample. In recent years, another method named DNA−protein interaction-ELISA (DPI-ELISA) has been developed to analyze TF activity.13−15 In this method, dsDNA probe is immobilized in microwell plates and an ELISA-like process was then executed to analyze TF activity. This method is suitable for analyzing a single TF in multiple protein samples in a high-throughput manner.16 However, because it depends on specific TF antibody, it cannot be used to analyze multiple TFs in a small amount of protein sample.17 Additionally, like antibodies used in supershift assay, the antibodies used in this method must bind the TF/DNA complex; however, most commercially available TF antibodies are manufactured just for the Western blot assay. The chromatin immunoprecipitation (ChIP)-grade TF antibodies can bind the TF/DNA complex, but they are the most expensive experimental materials for TF analysis.

NA-binding transcription factors (TFs) are a set of regulatory proteins that play important roles in gene expression regulation in various physiological and pathological processes.1 For example, NF-κB is one of important inducible TF that is ubiquitous in almost all cells.2 Dimers of NF-κB can bind κB sites in the genome to regulate the expressions of many target genes involved in many important biological processes, such as immune and inflammation.3 To TFs of this kind, analyzing their DNA-binding activity is indispensable to studying their functions. Therefore, the techniques for analyzing their activity have been highly valued. Electrophoretic mobility shift assay (EMSA) is the most widely used golden-standard method for analyzing TF activity.4,5 Autoradiography has been widely used to report EMSA signal.6 The major advantage of radioactive EMSA is its simpleness and high sensitivity.7 However, the isotope is dangerous to operators and the environment. Although chemiluminescent and fluorescent EMSA have been developed,8,9 researchers are still challenged by limited sensitivity, low throughput, and large sample consumption. Furthermore, to confirm the specificity of shifted DNA in EMSA detection of nuclear extract, a supershift assay that depends on a TF antibody that can bind TF proteins in the DNA/protein complex is still needed. Recently, several high sensitive methods based on DNA amplification have been developed, such as helicase-dependent amplification (HAD),10 exponential amplification reaction (EXPAR),11 and real-time polymerase chain reaction (PCR).12 These methods provide useful tools for sensitive analysis of TF activity. However, these methods depend on exonuclease to digest the remaining free DNAs before amplifying protein-bound © 2014 American Chemical Society

Received: November 19, 2013 Accepted: January 29, 2014 Published: January 29, 2014 2572

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Gene expression is coordinately regulated by various TFs.18 Therefore, analyzing multiple TFs can provide more valuable information for understanding the mechanism underlying gene expression regulation and the roles of various TFs in the cellular processes.18 For this end, several methods that can simultaneously analyze multiple TFs have been developed, such as the transignal protein/DNA array,19 the TF activation profiling plate array,20 the fluorescent microsphere-based multiplexed highthroughput assay,21 and the oligonucleotide array-based transcription factor assay (OATFA).22 These methods have already been used to profile the activities of many TFs in the particular protein sample23 and provide valuable tools for studying TFs. However, there are still difficulties in detecting the activities of TFs in trace amounts of protein samples due to their low sensitivity, which results from high interfering autofluorescence of the substrate in traditional visible fluorescence spectrum21,22 or colorimetric or chemiluminescent signal developments.19,20 Rolling circle amplification (RCA) is an isothermal DNA amplification technique that has high (linear RCA) or ultrahigh (multiprimed RCA) sensitivity.24 Due to its advantages of low constant reaction temperature (30 °C) and high sensitivity, it has been widely applied in bioanalysis.25 For example, RCA has been extensively used to detect viral DNA,26 DNA mutation,27 mRNA expression,28 and microRNA expression.29−31 The immunoRCA has been widely used to detect proteins.32,33 However, to our knowledge, this technique has still not been used to develop new methods for sensitively analyzing TF activity in high throughput format. Near infrared fluorescence (NIRF) has the exciting wavelength from 700 to 900 nm. In comparison with traditional visible fluorescence, NIRF has several significant advantages, including high sensitivity, high signal-to-noise (S/N) ratio, and deep tissue penetration capability.34−36 Therefore, NIRF techniques have been rapidly applied to in vitro assays of various biomolecules.37 For instance, NIRF-labeled antibodies and DNA probes were used to analyze interested proteins by Western blot,38 EMSA,39 DPI-ELISA,40 and in-cell Western.41 Furthermore, NIRF-labeled antibodies have been prevalently applied to in vivo molecular imaging of cancer biomarker proteins and photoimmunotherapy of cancers.42−44 Herein, we report a new assay for detecting TF activity that coupled solid-phase RCA (sRCA) with NIRF technique. This method was fully validated by detecting the DNA-binding activities of purified recombinant NF-κB p50 and NF-κB in HeLa cell nuclear extracts. The method was also enabled to simultaneously detect the DNA-binding activities of multiple TFs in HeLa cell nuclear extracts. This assay provides a new sensitive and high-throughput tool for studying TFs that can be used extensively in TF-related biological and biomedical research.

buffer were mixed in the same volume and heated for 5 min at 95 °C followed by slowly cooling to 25 °C. The mixture (5 μL) was ligated in a 25 μL ligation reaction, consisting of 14 units/μL T4 DNA ligase (TaKaRa) and 1 × T4 buffer (TaKaRa) at 37 °C for 30 min. The reaction was incubated at 65 °C for 5 min and run with agarose gel (2.5%) electrophoresis. The gel slice containing rolling circle DNA was cut and soaked in water (100 μL) overnight at 37 °C. The eluate was used as a rolling circle in NIRF-sRCA detection. Preparation of Capture Probe Array. The aminomodified oligonucleotides (Table S-1 of the Supporting Information) used as capture probe (CP) were dissolved in sterile water at the concentration of 20 μM and stored at 4 °C. The oligonucleotides were diluted with 50% dotting solution (CapitalBio) to a final concentration of 2 μM and spotted on aldehyde-modified glass slides (CapitalBio) with a spotting robot AD1500 (BioDot). The spotted slides were placed in a humidity chamber and incubated overnight at 37 °C. The slides were then washed with water for 2 min, 0.2% SDS for 2 min, and water for 2 min, respectively. Finally, the slides were bathed in 0.3% (w/v) NaBH4 for 5 min and washed three times with water. The slides were dried by spinning in a slide centrifuge (Labnet) and kept at 4 °C. Partition of Protein-Bound DNA. Protein-binding reaction (PBR) (10 μL) consisted of 1 × DNA-binding buffer (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 0.1 mM EDTA, 3 mM MgCl2, 1% glycerol, 0.5 mg/mL BSA, 0.05 mM DTT, 0.05% NP-40), 0.1 mg/mL PolydI-dC (Amershan), various amounts of purified recombinant NF-κB p50 proteins (Promega) or nuclear extracts (see figures), and 10 pmol TFBPs of each detected TF and NC. The reaction was kept at room temperature for 60 min. The reaction was mixed with 2 μL of 40% (v/v) glycerol and run with native polyacrylamide gel (PAGE) in 0.5 × TBE under 100 V at 4 °C for 1 h. After electrophoresis, gel was stained with 0.5 × TBE containing 0.5 μg/mL ethidium bromides (EB) (Amershan) for 30 min and the gel slice containing the shifted DNA was recovered under the UV transilluminator. The gel slice was soaked in 50 μL of diffusion buffer [0.5 M NH4AC, 10 mM Mg(AC)2, 1 mM EDTA, 0.1% SDS] overnight at 37 °C. The eluate was directly detected with NIRF-sRCA. Detection of Protein-Bound DNA with NIRF-sRCA. The eluates of rolling circle (5 μL) and the protein-bound DNA (8 μL) were mixed with 50 °C preheated hybridization solution (12.5 μL) (6 × SCC, 0.5% SDS, 5 × Denhardt’s solution, and 100 μg/μL herring sperm DNA) and added to the CP array. The CP array was covered with coverslip and incubated in a hybridization cassette (Arrayit) at 50 °C for 4 h. The slide was washed twice with distilled H2O for 2 min and spun dry. The RCA reaction (25 μL) that contained 10 units of φ29 DNA polymerase (New England BioLabs), 1 × φ29 buffer (New England BioLabs), 0.6 mM dNTP, and 0.012 mM Biotin-dUTP (Fermentas) was added to the CP array. The CP array was covered with a coverslip and incubated in an humid chamber at 30 °C for 30 min. The slide was washed twice with distilled H2O for 2 min. The slide was then successively incubated with blocking solution [maleic acid buffer containing 1% blocking reagents (Roche)] and IRDye 800CW-Streptavidin (LI-COR) (1:15000 diluted in maleic acid buffer) at room temperature for 1 h. Finally, the slide was washed twice in distilled H2O for 10 min and spun dry. The slide was imaged with an Odyssey Infrared Imaging System (LI-COR) at the channel of 800 nm (resolution: 21 and 84 μm; preset: membrane; quality: medium; focus offset: 3 mm; intensity: 6.0 to

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EXPERIMENTAL SECTION Preparation of TF Binding Probes and Rolling Circle. Oligonucleotides used in this study (Table S-1 of the Supporting Information) were synthesized using the standard phosphoramidate chemistry and were purified by high-performance liquid chromatography (HPLC) (Sangon). To prepare the TF binding probe (TFBP), the complementary oligonucleotides (100 μM) dissolved in the TEN buffer (10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA) were mixed in the same volume and heated for 5 min at 95 °C followed by slowly cooling to 25 °C. To prepare rolling circle, Oligo RC (100 μM) and RC link (200 μM) (Table S-1 of the Supporting Information) dissolved in TEN 2573

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Scheme 1. Schematic Illustration of the Procedures for Detecting the Activity of TFs with NIRF-sRCA

Table 1. CPs and TFBPs Used in This Study

and rolling circle to CP array, (iii) elongate the protein-bound TFBPs with sRCA that contains biotin-labeled dUTP, and (iv) detect the biotin-labeled sRCA products by binding of the NIRFlabeled streptavidin and NIRF imaging. To create CP arrays on glass surface modified with an aldehyde group, the oligonucleotides used as CPs were modified with an amino group at the 3′ end (Table 1). The sequences of CPs are complementary to their corresponding overhangs (tags) of TFBPs. TFBPs consist of three parts, including various overhangs complementary to CPs, double-stranded region containing TF binding sites (TFBSs) (underlined bases in Table 1) and constant overhang complementary to rolling circle (Table 1). A positive control probe (PC) was designed to monitor the CP coupling and NIRF-labeled streptavidin binding. PC was also used to normalize the signal. A negative control probe (NC) that contained no known TFBS was designed to

8.0). The signal was quantified with operating software of the Odyssey Infrared Imaging System. Preparation of Cell Extracts and Western Blot Detection. Culture and TNFα-stimulation of HeLa cells, preparation and quantification of nuclear extracts, and detection of nuclear extracts with Western blot were performed as previously described,45 and the detailed protocol was also provided in the Supporting Information. The NIRF-EMSA was performed as previously described,46 and the detailed protocol was also provided in the Supporting Information.

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RESULTS AND DISCUSSION Schematic Illustration of NIRF-sRCA Assay. NIRF-sRCA assay of TF activity is shown in Scheme 1. NIRF-sRCA detects TF activity in four steps: (i) incubate TFBPs with the detected protein sample such as nuclear extract and isolate protein-bound TFBPs via native PAGE, (ii) hybridize the protein-bound TFBPs 2574

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Figure 1. Determination of optimal CP concentration for creating CP arrays. (A) NIRF-sRCA detection of different amounts of NF-κB TFBPs with CP arrays fabricated with various concentrations of CP. (B) Quantified signal intensity of different arrays, according to CP concentration.

Figure 2. Detection of the activity of purified NF-κB p50 protein. (A) EB-stained PAGE gel loaded with PBRs. (B) NIRF image of NIRF-sRCA detection. PC and CP for NC TFBP were arrayed in triplex, and CP for NF-κB TFBP was arrayed in sixtuplex. (C) Quantified signal intensity.

monitor the detection specificity. PC and CP of NC were arrayed in triplex in each of CP arrays used for NIRF-sRCA assays. Establishment of sRCA System Containing BiotindUTP. Solid-phase RCA is a key step for the NIRF-sRCA assay. We first determined the optimal dosage of biotin-dUTP in liquid RCA reaction and found that a liquid RCA reaction containing 12 μM of biotin-dUTP obtained similar amplification efficiency as the same liquid RCA system without biotin-dUTP (Figure S-1A of the Supporting Information). This RCA formula (dTTP:biotin-dUTP = 50:1) was then applied to sRCA. We found that it produced high RCA efficiency and NIRF signal in NIRF-sRCA detection (Figure S-1B of the Supporting Information). This optimized sRCA system was thus employed in all subsequent NIRF-sRCA detections.

To check the specificity of the NIRF-sRCA detection, we constructed CP arrays with PC and CPs for NC and NF-κB TFBPs. The fabricated CP arrays were incubated with the hybridization solutions that contained one or both of NC and NF-κB TFBPs and detected with the optimized NIRF-sRCA conditions. The results revealed that the NIRF-sRCA specifically detected the target DNAs (Figure S-2 of the Supporting Information). NIRF-sRCA assay adopted biotin−streptavidin and IRDye800CW because of their advantages. Biotin−streptavidin has the strongest binding affinity and wide application in biological assays.47 IRDye800CW is a NIRF fluorophore with the maximum exciting/emission wavelength of 785/810 nm. We found that the solid support of the glass slide showed almost no 2575

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Figure 3. Detecting NF-κB activity in HeLa cell nuclear extracts. (A) EB-stained PAGE gel loaded with PBRs and NIRF-sRCA detection of proteinbound DNA. Nuclear extracts (NE) in PBRs: 1, 10 μg (TNFα+); 2, 5 μg (TNFα+); 3, 0 μg; 4, 10 μg (TNFα+); 5, 10 μg (TNFα+) plus 50 pmol competitor; 6, 0 μg; 7, 10 μg (TNFα+); 8, 10 μg (TNFα-); 9, 0 μg. TNFα+, TNFα induced; TNFα-, TNFα uninduced. CP layout is the same as Figure 2. (B) Quantified signal intensity. (C) Western blot assay of NF-κB in nuclear extracts. FI = fluorescence intensity.

detectable background fluorescence at the exciting wavelength of this dye. Therefore, this dye showed the highest S/N ratio in NIRF-sRCA assays. Optimization of CP Concentration for Preparing CP Array. To determine the optimal concentration of CP for preparing DNA arrays and to check the sensitivity of the NIRFsRCA assay, we fabricated CP arrays by spotting NF-κB CP at various concentrations (0.125, 0.5, and 2 μM) and performed NIRF-sRCA assays of various amounts of NF-κB TFBPs. The results revealed that the CP concentration of 2 μM produced the best detection signal and linear correlation between the NIRF signal and the amount of target (Figure 1). At this CP concentration, the detection limit of the NIRF-sRCA assay reached 10 fmol of targets and a linear correlation with R2 > 0.98 was obtained in the range of 0 to 50 fmol of targets. Therefore, we created all CP arrays with this optimized CP concentration for all subsequent NIRF-sRCA detection. Detection of Purified Recombinant NF-κB p50 Protein. When the optimal conditions for the NIRF-sRCA assay were determined, we first detected the DNA-binding activity of purified recombinant NF-κB p50 protein with this assay. The same amounts (10 pmol) of NF-κB and NC TFBPs were reacted with six different amounts of p50 proteins and run with native PAGE. The NF-κB-bound TFBPs were then detected with NIRF-sRCA. The results demonstrated that the NIRF signal intensity increased with the p50 protein amount in PBR (Figure 2). The negative control PBR that contained no p50 protein produced no NIRF signal (Figure 2). The features of NC CPs also showed no signal despite the presence of NC TFBPs in all PBRs (Figure 2). These results demonstrate that the NF-κB p50

activity in the detected protein sample was quantitatively and specifically detected by NIRF-sRCA. The results also demonstrate that as little as 6.94 ng (∼140 fmol) of the p50 protein was detected by NIRF-sRCA. In addition, NIRF-sRCA is more sensitive than NIRF-EMSA and EB-EMSA (Figure S-3 of the Supporting Information). In this study, we used native PAGE to partition the proteinbound TFBPs; however, other partition approaches, including filter binding48 and agarose gel electrophoresis,49,50 can also be employed to isolate protein-bound TFBPs. Especially, in place of native PAGE with filter binding, the NIRF-sRCA assay can be finished in 8 h. In addition, a new rapid DNA elution method that we just recently developed can also be employed to elute DNAs from PAGE gels in as little time as 5 min (Figure S-4 of the Supporting Information). With the rapid elution method, the NIRF-sRCA assay can be finished in 9 h. Detection of NF-κB Activity in HeLa Cell. We next detected the NF-κB activity in a bona fide protein sample, HeLa cell nuclear extract, with NIRF-sRCA. To preliminarily detect nuclear extracts, two PBRs contained 10 and 5 μg of TNFαinduced HeLa cell nuclear extracts were detected with NIRFsRCA. The results revealed that the NIRF signal increased with the amounts of nuclear extracts in PBRs (Figure 3, panels A and B, PBR1 and 2). To confirm the specificity of NF-κB detection with nuclear extracts, two PBRs with equal amounts of TNFαinduced HeLa cell nuclear extracts (10 μg), but one of them with excess competitive dsDNA (50 pmol), were detected with NIRFsRCA. The results showed that the addition of competitive dsDNA significantly reduced the NIRF signal (Figure 3, panels A and B, PBR4 and 5). To further confirm the specificity of NF-κB 2576

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Figure 4. Detecting NF-κB activity in various amounts of nuclear extracts. (A) EB-stained PAGE gel loaded with PBRs and NIRF-sRCA detection of protein-bound DNA. CP layout is the same as Figure 2. (B) Quantified signal intensity. (C) Western blot assay of NF-κB in nuclear extracts. FI = fluorescence intensity.

Figure 5. Mock detection of multiple TFs. (A) NIRF-sRCA detection of TFBP samples. RCA reactions: reactions 1 and 2 contained various amounts of different TFBPs, and reaction 3 contained the same amounts of different TFBPs. PC and CPs were arrayed in triplex. CP layout in array: top four columns from left to right: NF-E2, AP1, NC and PC. Bottom four columns from left to right: NF-κB, TFIID, p53 and CREB. (B) Quantified signal intensity. FI = fluorescence intensity.

detection with nuclear extracts, two PBRs that contained equal amounts (10 μg) of nuclear extracts from TNFα-induced and -uninduced HeLa cells, respectively, were detected with NIRFsRCA. The results demonstrated that the TNFα treatment increased the NF-κB activity in HeLa cells (Figure 3, panels A and B, PBR7 and 8), which was in accordance with the Western blot assay (Figure 3C). Finally, to find the limit of NIRF-sRCA detection of the nuclear extract, five PBRs containing various amounts of TNFα-induced HeLa cell nuclear extracts were detected with NIRF-sRCA. The result revealed that the NF-κB activity in as little as 0.625 μg TNFα-induced HeLa cell nuclear extracts was detected by NIRF-sRCA (Figure 4). In all above nuclear extract detections, a negative control PBR that contained

no nuclear extract was included; however, these negative control PBRs did not produce signals (Figure 3, PBR3, 6 and 9; Figure 4, PBR6). It should be pointed out that we included polydI-dC in all PBRs that were used to detect the nuclear extract in order to eliminate the possible nonspecific binding of TFBPs with nontarget proteins. Because polydI-dC bound EB and thus produced smears in lanes of PAGE gels, the gels between loading wells and free DNA bands (see figures) were cut for recovering NF-κB-bound TFBPs. Detection of Activities of Multiple Transcription Factors in Parallel. Finally, we detected activities of multiple TFs in a bona fide protein sample, HeLa cell nuclear extract, with 2577

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Figure 6. Detection of activities of multiple transcription factors in HeLa cells. (A) PBRs and EB-EMSA. (B) NIRF-sRCA detection of protein-bound DNA. CP layout in array is the same as Figure 5. (C) Quantified signal intensity. (D) TFIID-normalized activities of multiple TFs detected with NIRFsRCA and TranSignal Protein/DNA Array I (P/D Array I). (E) Fold induction of TF activity by TNFα in HeLa cells. (F) Correlation analysis of fold induction detected with NIRF-sRCA and TranSignal Protein/DNA Array I (P/D Array I).

specific CP and TFBP can be designed for all of about 700 DNAbinding TFs in the human genome, this method can be scalable to profile the genome-wide activities of DNA-binding TFs. With the maximum multiplex of detected targets, the activities of many TFs in a small amount of protein sample can be detected with this method. Several other advantages of sRCA also benefit this method. Solid-phase RCA combines amplification and isolation of target DNA together, which simplifies the detection process. In NIRF-sRCA detection, RCA products were easily isolated from the RCA reaction by a brief washing of slides. RCA amplifies target DNAs at low constant temperature (30 °C), which is helpful for carrying out NIRF-sRCA detection without relying on special instruments. Finally, the high sensitivity of RCA contributes to the high sensitivity of NIRF-sRCA. This study demonstrates that as little as 10 fmol of TFBP, 6.94 ng (about 140 fmol) of purified NF-κB p50 protein, and 0.635 μg of nuclear extract can be detected by NIRF-sRCA. Because only about one-sixth (8 μL of 50 μL) of gel eluate that contained protein-bound DNA was detected with NIRF-sRCA, the true sensitivity of this method for detecting TF activity should be higher than that described here.

NIRF-sRCA. For this end, we designed specific CPs and TFBPs for five other TFs (Table 1), including AP-1, TFIID, CREB, NFE2, and p53 and fabricated CP arrays. To check the specificity of arrayed CPs to their target TFBPs, single TFBP was added in a hybridization solution and detected with NIRF-sRCA. The results revealed that all CPs were specific to their target TFBPs (Figure S-5 of the Supporting Information). To further check the specificity of CPs and the capability of NIRF-sRCA for a quantitative assay of relative abundance of TFBPs in the detected DNA sample, three DNA samples that were composed of various amounts of multiple TFBPs were added in a hybridization solution and detected with NIRF-sRCA. The results revealed that the NIRF-sRCA accurately detected the relative abundance of various targets in the detected DNA sample (Figure 5). Then, we detected the activities of six TFs in the nuclear extracts of TNFα-induced and -uninduced HeLa cells with NIRF-sRCA. A negative control PBR and two PBRs containing equal amounts (10 μg) of nuclear extracts obtained from TNFαinduced and -uninduced HeLa cells, respectively, were detected with NIRF-sRCA. All PBRs contained 10 pmol of TFBPs of each detected TFs and NC. The results were shown in Figure 6. By using TFIID as an internal control as in the Western blot assay of nuclear protein,51 we normalized the signals of five TFs with that of TFIID and calculated the fold induction of these TFs. The results revealed that the TNFα inducement increased the activity of NF-κB (2.39 fold), CREB (1.69 fold), and AP1 (1.7 fold) in HeLa cells, which is in accordance with the previous TranSignal Protein/DNA array I detection results.50 The fold inductions of these TFs analyzed with two methods strongly correlated (R2 = 0.73; Pearson’s r = 0.83). The major advantage of sRCA is that it enables NIRF-sRCA to analyze activities of multiple TFs simultaneously. Given that the

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CONCLUSION This study developed an antibody-free method for analyzing TF activity that combined sRCA with the NIRF technique. With the optimized assaying conditions, the feasibility and reliability of this method were fully demonstrated by detecting the DNAbinding activities of purified recombinant NF-κB p50 and NF-κB in HeLa cell nuclear extracts. The scalability of this method was also revealed by detecting the DNA-binding activities of six TFs in TNFα-induced and -uninduced HeLa cell nuclear extracts. This study thus provides a new high sensitive method for 2578

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Analytical Chemistry

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analyzing DNA-binding activities of TFs. This method can easily be adapted to analyze the activities of a single TF in multiple protein samples or multiple TFs in a protein sample in the highthroughput format.

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ASSOCIATED CONTENT

S Supporting Information *

Experimental Section and Figures S-1 to S-5. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +86 25 83793620. Fax: +86 25 83793620. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the grants from the National Natural Science Foundation of China (Grant 61171030) and the Technology Support Program of Jiangsu (Grant BE2012741).

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REFERENCES

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dx.doi.org/10.1021/ac403758p | Anal. Chem. 2014, 86, 2572−2579