Sensitive Detection of Transcription Factor in Nuclear Extracts by

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Sensitive Detection of Transcription Factor in Nuclear Extracts by TargetActuated Isothermal Amplification-Mediated Fluorescence Enhancement Yan Zhang, Dong-xue Xiang, Bo Tang, and Chun-yang Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02451 • Publication Date (Web): 28 Aug 2017 Downloaded from http://pubs.acs.org on August 28, 2017

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Analytical Chemistry

Sensitive Detection of Transcription Factor in Nuclear Extracts by Target-Actuated Isothermal Amplification-Mediated Fluorescence Enhancement Yan Zhang,† Dongxue Xiang,† Bo Tang* and Chun-yang Zhang*

College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, Shandong Normal University, Jinan 250014, China. * Corresponding author. Tel.: +86 0531-86186033; Fax: +86 0531-82615258. E-mail: [email protected].:+86

0531-86180010;

Fax: +86

[email protected].

1

ACS Paragon Plus Environment

0531-86180017;


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ABSTRACT: Transcription factors (TFs) modulate the process of gene transcription by binding to specific DNA sequences, and their alteration may cause a variety of diseases. Here, we develop a simple and sensitive method to directly detect TF in crude nuclear extracts using target-actuated isothermal amplification-mediated fluorescence enhancement with fluorescent base 2-aminopurine (2-AP) as the fluorophore. In the presence of TF, its specific binding to the probe prevents the digestion of the probe by Exonuclease III (Exo III), initiating the extension reaction to produce DNA duplexes which may be subsequently digested by lambda exonuclease to release single-stranded DNAs (ssDNAs) and free 2-AP molecules. Although some excess probes may be partially digested by Exo III, the phosphorothioate modification between two binding sites of the probe may generate a hindrance to preserve the rest of TF-binding probes which may hybridize with the released ssDNAs to initiate new cycles of nicking-digestion-hybridization, generating abundant free 2-AP molecules for significant fluorescence enhancement. Different from the reported amplification strategies, all the TF-binding probes take part in the amplification reaction no matter they bind with TF or not, greatly improving the detection signal. This method can be used for sensitive detection of NF-κBp50 with a detection limit of 4.11 × 10-4 mg/mL and the screening of potential TF inhibitors as well. Importantly, this method is very simple without the involvement of any external quenchers, extra primers and templates, and it may be extended to selectively detect various DNA-binding proteins by simply changing the binding-site sequences of the probes.

2


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INTRODUCTION Transcription factors (TFs) are a group of sequence-specific DNA-binding proteins, which modulate the process of gene transcription by binding to specific double-stranded DNA (dsDNA) sequences.1 In human genomes, about 1500 TF-coding genes regulate waves of TF production and activation, and play important roles in cell proliferation, differentiation and growth.2,3 Conversely, alteration of TF level may lead to a variety of diseases including cancers,4,5 developmental disorders,6 abnormal hormone responses,7,8 autoimmunity and inflammation.9,10 Thus, sensitive detection of TFs is of great importance to both biomedical researches and clinical diagnosis.11 Traditional methods for TF assay include electrophoretic mobility shift assay (EMSA),12 DNA footprinting assays,13 enzyme-linked immunosorbent assay (ELISA),14 and Western blotting assay,15 but they are usually laborious and time-consuming12,13 with the requirement of radioisotope labels and specific antibodies.14,15 Alternatively, fluorescent methods have been developed for TF assay with the advantages of safety, simplicity and high sensitivity.16-19 Typical examples include the use of molecule beacon as an efficient signal-transduction platform for TF assay, but the involvement of complicated procedure in labelling the DNA probe with two fluorochromes16-19 and the rational design of a covalent break within the TF binding site may inevitably increase the experimental cost and complexity.16,17 To improve the detection sensitivity, a variety of nucleic acid amplification strategies have been introduced, such as real-time polymerase chain reaction (PCR),20,21 exponential amplification reaction (EXPAR),22,23 helicase-dependent amplification (HDA),24 rolling circle amplification (RCA),25 and near-infrared fluorescent solid phase-based RCA (NIRF-sRCA) assay.26 Among these 3



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approaches, the real-time PCR has gained increasing attention due to its high sensitivity and practicality, but it requires the precise control of temperature cycling with the involvement of TaqMan probes and specific antibody-DNA conjugates.20,21 Although EXPAR may provide a high amplification efficiency within minutes, it involves complicated reaction schemes.22,23 The EXPAR-based colorimetric assay needs the modification of gold nanoparticles,22 and the EXPAR-induced chemiluminescence assay involves DNA transcription, dual EXPAR reaction and G-quadruplex DNAzyme-driven chemiluminescence reaction.23 The NIRF-sRCA assay involves the careful partition of protein-bound DNA probes and the complicated preparation of capture probes and rolling circle templates.26 Notably, all these amplification strategies require extra primers and/or templates to initiate the nucleic acid amplification, and thus the special design of primers and/or templates is necessary.20-26 For example, real-time PCR and HAD require specific priemrs;20,21,24 EXPAR needs specially designed templates;22,23 RCA requires both a dumbbell-shaped template and a primer;25 the NIRF-sRCA assay needs a rolling circle template.26 Therefore, it is necessary to develop a simple and cost-effective method for sensitive detection of TFs in crude nuclear extracts without the involvement of any separation steps, external quenchers and complicated design of primers and templates. In this research, we develop a sensitive fluorescent method to directly detect transcription factor

NF-κB

p50

in

crude

nuclear

extracts

using

target-actuated

isothermal

amplification-mediated fluorescence enhancement with fluorescent base 2-aminopurine (2-AP) as the fluorophore without the involvement of any separation steps, external quenchers, extra primers and templates. In contrast to the conventional fluorescent probes, the quenching of 2-AP fluorescence requires no extra quenchers and the 2-AP may be attached at any position 4


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of the probes.27-29 The 2-AP exhibits a weak fluorescence when being incorporated into the dsDNA, but an enhanced fluorescence when being free in the solution. Due to the low background signal resulting from efficient stacking interaction of 2-AP with the bases of TF-binding probes, the involvement of both bound and unbound TF-binding probes in the amplification reaction and exonuclease-assisted recycling signal amplification, the proposed method can sensitively detect endogenous NF-κB p50 in crude nuclear extracts with a detection limit of 4.11 × 10-4 mg/mL, and it can be applied for the screening of potential TF inhibitors as well.

EXPERIMENTAL SECTION Materials. The oligonucleotides (Table 1) and deoxynucleotide solution mixture (dNTPs) were obtained from Takara Biotechnology Co. Ltd. (Dalian, China). The Exonuclease III (Exo III), Klenow Fragment polymerase (3'→5' exo-, KF polymerase), lambda exonuclease and the Nb.BtsI nicking endonuclease were purchased from New England Biolabs (Beverly, MA, USA). The SYBR Gold was purchased from Life Technologies (Carlsbad, CA, USA). Magnesium chloride (MgCl2), ethylenediaminetetraacetic acid (EDTA), trizma hydrochloride (Tris-HCl, pH 8.0) and sodium chloride (NaCl) were obtained from Sigma-Aldrich Company (St. Louis, MO, USA). Oridonin was obtained from Shanghai Macklin Biochemical Company (Shanghai, China). Ultrapure water was prepared by a Millipore filtration system (Millipore, Milford, MA, USA). Table 1. Sequences of the Oligonucleotidesα note

sequence (5′-3′) 5



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p50-s

A*A*A ACT↓ CAC TGC ACT CGA TCG GAC AGA TGG GAC TTT CCT TGA TA*G* GAC CTG GGG GAC TTT CCA ATA

p50-anti

A*A*A ACT↓ CAC TGC ACT CGA TCG GAC TAT TGG AAA GTC CCC CAG GT*C* CTA TCA AGG AAA GTC CCA TCT

p50-s mt

A*A*A ACT↓ CAC TGC ACT CGA TCG GAC AGA TCTCAC TTT CCT TGA TA*G* GAC CTG GCTCAC TTT CCA ATA

p50-anti mt

A*A*A ACT↓ CAC TGC ACT CGA TCG GAC TAT TGG AAA GTGAGC CAG GT*C* CTA TCA AGG AAA GTGAGA TCT

assistant probe α

T*TC GAT CGA GTG CA*C*A-NH2

The asterisks indicate the phosphorothioate modification. The boldface bases represent the

binding region of NF-κB p50. The underlined italic bases indicate the 2-aminopurine substitution. The italic regions of p50-s and p50-anti probes indicate the binding sequence of assistant probe. The italic region of assistant probe indicates the binding sequences of p50-s and p50-anti probes. The arrows in the probes indicate the nicking positions of Nb.BtsI. The underlined bold letters symbolize the mutant bases in the binding site of NF-κB p50.

Cell Culture and Preparation of Nuclear Extracts. HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, gibco, USA) with 10% fetal bovine serum (FBS, gibco, USA) and 1% penicillin-streptomycin (Invitrogen, USA) at 37 °C with 5% CO2. The HeLa cells were incubated with 20 ng/mL TNF-α (Invitrogen, USA) for 30min. The nuclear extracts were collected using a nuclear extract kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer’s instructions, and the concentration of proteins was measured using 6


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Bradford-based assay. Protein-DNA Interaction and Exonuclease III Digestion. For the preparation of TF-binding probes, 10 µM p50-s probes, 10 µM p50-anti probes and 20 µM assistant probes were incubated in a buffer containing 10 mM Tris-HCl (pH 8.0), 100 mM NaCl at 95°C for 5 min, followed by slowly cooling to room temperature. The obtained DNA stock solutions were stored at ‒20°C for further use. The 1 µL of nuclear extracts at various concentrations and 1 µM TF-binding probes were incubated at 37 °C for 30 min in 10 µL of binding buffer containing 10 mM Tris-HCl (pH 7.5), 100 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 0.1 mg/mL yeast tRNA, 10% glycerol, 0.25 mM DTT. Then 10 units of Exo III and 1 µL of 10× NEB buffer 1 were added to the mixture and incubated for another 10 min at 37 °C. The digestion reaction was terminated by heating at 80°C for 10 min. The SYBR Gold with the final concentration of 1 µg/mL was added into the samples, and then the mixture was loaded on a 10% nondenaturing polyacrylamide gel and electrophoresed at 110 V in 1× Tris-borate-EDTA (TBE) buffer. The image of gel electrophoresis was visualized by a ChemiDoc MP Imaging system (Hercules, California, USA). Fluorescence Measurement of NF-κB p50. The experiments were performed in 20 µL of solution containing 2 µL of digestion mixture, 250 µM dNTPs, 1 U KF polymerase, 1 U Nb.BtsI, 1 U lambda exonuclease, 1× NEBbuffer 2 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM DTT), 1× NEB Lambda buffer (67 mM Glycine-KOH, 2.5 mM MgCl2, 50 µg/ml BSA), and incubated at 37 °C for 50 min. The 20 µL of reaction products was diluted to a final volume of 60 µL with ultrapure water. The fluorescence spectra were measured with a Hitachi F-7000 fluorescence spectrophotometer (Tokyo, Japan) at an excitation wavelength 7



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of 310 nm, and the fluorescence intensity at 365 nm was recorded for data analysis. The Inhibition Assay. For transcription factor inhibition assay, 20 µM oridonin was preincubated with 0.25 mg/mL nuclear extracts and 1 µM TF-binding probes at 37 °C for 30 min in 10 µL of binding buffer. The subsequent reactions followed the above steps, and fluorescence intensity of 2-AP was measured as described above.

RESULTS AND DISCUSSION Principle of Transcription Factor Assay. The principle of TF assay in illustrated in Scheme 1. The linear TF-binding probe consists of a p50-s probe and a p50-anti probe, which bear two specific TF recognition elements (Scheme 1, red color). The 5′ ends of p50-s probe and p50-anti probe contain the same sequence with two substitutions of 2-AP molecules for adenines. To reduce the background fluorescence signal, the TF-binding probe is designed to be complementary to the assistant DNA probe (Scheme 1, blue color). In addition, the phosphorothioate modification (Scheme 1, asterisks) may efficiently prevent the nonspecific digestion of the probes by exonuclease.30 In this research, the NF-κB p50-initiated amplification reaction may be performed in one tube, with all the TF-binding probes taking part in the amplification reaction no matter they bind with TF or not (Scheme 1). In the presence of NF-κB p50, the binding of TF-binding probes with p50 can initiate the first-step amplification reaction (Scheme 1, right circle), whose product may subsequently react with the free TF-binding probes to initiate the second-step amplification (Scheme 1, right circle), thus greatly amplifying the detection signal. In the first-step amplification reaction, the binding of NF-κB p50 with the linear 8


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TF-binding probe may prevent the digestion of the probe by exonuclease III (Exo III). The 3′ end of the TF-binding probe may function as the primer to initiate the extension reaction simultaneously in both directions in the presence of polymerase, producing a DNA duplex with the recognition site (Scheme 1, green color) for Nb.BtsI nicking enzyme. The subsequent cleavage of DNA duplex by Nb.BtsI nicking enzyme may yield the site of phosphate group (PO4) for lambda exonuclease which may catalyze the removal of 5′ mononucleotides from the duplex DNA in 5′ to 3′ direction. Notably, the phosphorothioate modification between the two binding sites of TF-binding probe may generate a hindrance to preserve the rest of the probe,30 which is subsequently dissociated to release 2-AP molecules and new ssDNA. Unlike the reported TF assays in which the excess detection probes are completely digested by Exo III,20,22-24 in this assay the excess TF-binding probes are protected from the Exo III digestion due to the steric hindrance induced by phosphorothioate modification.30 The released ssDNA may hybridize with the rest of p50-s probe and p50-anti probe, respectively, to form the complete DNA duplexes with the recognition sites for Nb.BtsI nicking enzyme, subsequently initiating the second-step amplification reaction to release abundant free 2-AP molecules and ssDNAs. Notably, the ssDNAs produced from the first-step amplification reaction may hybridize with the p50-s probes and the p50-anti probes in the second-step amplification reaction to initiate the cycles of nicking-digestion-hybridization, releasing abundant free 2-AP molecules for enhanced fluorescence signal. While in the absence of the TF (Scheme 1, left circle), TF-binding probe will be degraded by Exo III, resulting in the dissociation of the p50-s-p50-anti hybrid. Thus, neither extension reaction nor digestion reaction may occur, and no significant fluorescence enhancement is observed. 9



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Scheme 1. Schematic illustration of TF assay in nuclear extracts on the basis of target-actuated isothermal amplification-mediated fluorescence enhancement with fluorescent base 2-AP as the fluorophore. In the presence of NF-κB p50 (right circle), the binding of TF-binding probes with p50 can initiate the first-step amplification reaction, whose product may subsequently react with the free TF-binding probes to initiate the second-step amplification, greatly amplifying the detection signal. While in the absence of the TF (left circle), the digestion of TF-binding probe by Exo III abolishes the subsequent two-step amplification reaction, and no significant fluorescence enhancement is observed.

Feasibility Study. In this research, the treatment of HeLa cells with TNF-α was used to increase the amount of NF-κB,31 and the gel electrophoresis was employed to analyze the digestion of TF-binding probe by Exo III (Figure 1A). In the presence of nuclear extracts, the binding of NF-κB p50 with the TF-binding probes protects the probes from being digested by Exo III. In contrast, the naked probes without NF-κB p50 binding will be partially degraded by Exo III, with the rest of TF-binding probe being preserved due to the steric hindrance 10


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induced by the phosphorothioate modification between two binding sites (Scheme 1). As a result, two distinct DNA bands including the TF-binding probes and the partially digested probes are observed in the presence of nuclear extracts (Figure1A, lane 1), while only one band of partially digested probe is observed in the absence of nuclear extracts (Figure 1A, lane 2). We further used the fluorescence measurement to verify the feasibility of the proposed assay (Figure 1B). In the presence of nuclear extracts, a strong 2-AP fluorescence signal with the characteristic emission peak of 365 nm is observed (Figure 1B, red line). However, no significant fluorescence signal is observed in the presence of inactivated nuclear extracts (Figure 1B, blue line) and in the control groups without nuclear extracts (Figure 1B, black line). These results indicate that the binding of NF-κB p50 with the TF-binding probes can protect the probes from being digested by Exo III and subsequently initiates the two-step amplification reaction for enhanced fluorescence signal. Notably, we performed all experiments (including the subsequent optimization of experimental conditions) using cell nuclear extracts. This has never been achieved by any previous publications, suggesting that our method is solid and ready for real sample analysis.

Figure 1. (A) Nondenaturating PAGE analysis of the TF-binding probe digested by Exo III in the presence (lane 1) and absence of nuclear extracts (lane 2). Lane 1 represents 10 µg of 11



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nuclear extracts + 200 nM NF-κB p50-specific probes, lane 2 represents 200 nM NF-κB p50-specific probes without nuclear extracts. (B) Fluorescence emission spectra of reaction products in the presence of 5.5 mg/mL nuclear extracts (red line), 5.5 mg/mL inactivated nuclear extracts (blue line), and in the absence of nuclear extracts (black line).

Optimization of Experimental Conditions. This assay involves three kinds of enzymes and three corresponding buffers (i.e., CutSmart buffer, NEBbuffer 2 and lambda buffer). The NEBbuffer 2 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM DTT) is for KF polymerase, the CutSmart buffer (50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 µg/ml BSA) is for Nb.BtsI, and 1× NEB Lambda buffer (67 mM glycine-KOH, 2.5 mM MgCl2, 50 µg/ml BSA) is for lambda exonuclease. Because the Nb.BtsI has high activity in both CutSmart buffer and NEBbuffer 2,32 and KF polymerase has 100% activity in NEBbuffer 2. Therefore, NEBbuffer 2 should be a necessary buffer in the experiments. To obtain the optimal reaction buffer, we investigated the performance of the proposed assay in CutSmart buffer + NEBbuffer 2, lambda buffer + NEBbuffer 2 and the mixture buffer (CutSmart buffer: NEBbuffer 2: lambda buffer = 1:1:1), respectively. As shown in Figure 2A, the value of (F-F0)/F0 in response to lambda buffer + NEBbuffer 2 is much higher than those in response to CutSmart buffer + NEBbuffer 2 and the mixture buffer, where F and F0 are the fluorescence intensity in the presence and absence of nuclear extracts, respectively. Thus, lambda buffer + NEBbuffer 2 is used in the subsequent research. The signal amplification relies on the cooperation of nicking endonuclease and lambda exonuclease, and thus the amount of Nb.BtsI and lambda exonuclease should be carefully 12


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optimized. We investigated the influence of lambda exonuclease upon the fluorescence signal with a fixed amount of Nb.BtsI (2 U). As shown in Figure 2B, the maximum value of (F-F0)/F0 is obtained at 1 U lambda exonuclease (F and F0 are the fluorescence intensity in the presence and absence of nuclear extracts, respectively). Therefore, 1 U lambda exonuclease is used in the subsequent research. We further investigated the influence of Nb.BtsI upon the fluorescence signal with a fixed amount of lambda exonuclease (1U). As shown in Figure 2C, the value of (F-F0)/F0 increases with the increasing amount of Nb.BtsI from 0.1 to 1 U, followed by the decrease beyond the amount of 1 U (F and F0 are the fluorescence intensity in the presence and absence of nuclear extracts, respectively). Thus, 1 U Nb.BtsI is used in the subsequent research. We investigated the influence of reaction time upon the fluorescence signal as well. As shown in Figure 2D, the value of (F-F0)/F0 increases with the reaction time from 10 to 50 min and reaches a plateau at 50 min (F and F0 are the fluorescence intensity in the presence and absence of nuclear extracts, respectively). Thus, the reaction time of 50 min is used in the subsequent research.

13



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Figure 2. (A) The value of (F-F0)/F0 in response to the mixture buffer, CutSmart buffer + NEBbuffer 2, and lambda buffer + NEBbuffer 2, respectively. (B) The value of (F-F0)/F0 in response to different amount of lambda exonuclease at a fixed amount of Nb.BtsI (2 U). (C) The value of (F-F0)/F0 in response to different amount of Nb.BtsI at a fixed amount of lambda exonuclease (1 U). (D) Variance of (F-F0)/F0 value with reaction time. F and F0 are the fluorescence signals in the presence and absence of nuclear extracts, respectively. The concentration of nuclear extracts is 0.55 mg/mL. Error bars show the standard deviation of three experiments.

Detection Sensitivity. To demonstrate the improved sensitivity of the proposed method for NF-κB p50 assay in HeLa cell nuclear extracts, we measured the fluorescence intensity in response to different-concentration nuclear extracts under the optimized experimental conditions. Figure 3A shows the variance of fluorescence emission spectra with the 14


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concentration of nuclear extracts. The fluorescence intensity at the emission wavelength of 365 nm increases with the increasing concentration of nuclear extracts from 5.5 × 10-4 to 5.5 mg/mL. In logarithmic scales, the fluorescence intensity exhibits a linear correlation with the concentration of nuclear extracts through a detection range over 4 orders of magnitude from 5.5 × 10-4 to 5.5 mg/mL (Figure 3B). The regression equation is F = 453.33 + 113.16 log10 C (R2 = 0.993), where F is the fluorescence intensity and C is the concentration of nuclear extracts, respectively. The detection limit is calculated at 4.11 × 10-4 mg/mL on the basis of the average signal of blank plus three times standard deviation. The sensitivity of the proposed method has improved by 2 orders of magnitude compared with those of NIRF-sRCA assay (6.25 × 10-2 mg/mL)26 and the reported FRET assay (5 × 10-2 mg/mL),18 and it is comparable to that of real-time PCR assay (5 × 10-4 mg/mL).20 The improved sensitivity might be ascribed to (1) the low background signal resulting from efficient stacking interaction of 2-AP with the bases of TF-binding probes,28,29 (2) the involvement of both bound and unbound TF-binding probes in the amplification reaction, and (3) the efficient signal amplification induced by lambda exonuclease-catalysed multiple turnover reactions. Notably, in comparison with the reported methods for TF assay (Table 2), the proposed method is extremely sensitive and convenient without the involvement of any separation steps, external primers, extra templates and antibodies.

15



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Figure 3. (A) Fluorescence emission spectra in response to different-concentration nuclear extracts. (B) Linear relationship between the fluorescence intensity at 365 nm and the logarithm of nuclear extract concentration in the range from 5.5 × 10-4 to 5.5 mg/mL. Error bars show the standard deviation of three experiments.

Table 2. Comparison of the Proposed Methods with the Reported Methods for TF Assay. method/technique

involvement of extra primers, templates or antibodies

requirement of separation steps

detection limit

references

target-actuated isothermal amplification-mediated fluorescence enhancement

no

no

nuclear extracts, 4.11 × 10-4 mg/mL

this work

real-time PCR

specific primers

no

nuclear extracts, 5×

20

16


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10-4 mg/mL near-infrared fluorescent solid phase-based RCA (NIRF-sRCA)

rolling circle templates

yes

exponential amplification reaction (EXPAR)-based colorimetric assay EXPAR-induced chemiluminescence assay

specific templates

no

specific templates

no

helicase-dependent amplification (HDA)

specific primers

no

enzyme-linked immunosorbent assay (ELISA) fluorescence resonance energy transfer (FRET) assay

specific antibody

yes

no

no

recombinant NF-κB, p50 1.4 × 10-8 M, nuclear extracts, 6.25 × 10-2 mg/mL recombinant NF-κB p50, 3.8 × 10-12 M recombinant NF-κB p50, 6.03 × 10-15 M recombinant NF-κB p50, 9.3 × 10-13 M nuclear extracts, 1 × 10-2 mg/mL recombinant NF-κB p50, 5 × 10-9 M, nuclear extracts, 5 × 10-2 mg/mL

26

22

23

24

14

18

Detection Specificity. To investigate the specificity of the proposed method for NF-κB p50 assay, we used an irrelevant protein of bovine serum albumin (BSA) and a nonspecific detection probe (Table 1, p50-s mt and p50-anti mt) with the sequence of CTC ACT TTC C in place of the p50 binding sequence of GGG ACT TTC C (Table 1, p50-s and p50-anti). As shown in Figure 4, a high value of (F-F0)/F0 (F and F0 are the fluorescence intensity in the presence and absence of protein, respectively) is observed only in the presence of nuclear extracts and specific binding probes (Figure 4, red column), while an extremely low value of (F-F0)/F0 is observed in response to either a nonspecific probe (Figure 4, blue column) or an 17



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irrelevant protein of BSA (Figure 4, green column). These results clearly demonstrate the high specificity of the proposed method for NF-κB p50 assay even in TNF-α-induced HeLa cell nuclear extracts.

Figure 4. Specificity of the proposed method for NF-κB p50 assay in nuclear extracts. The tested samples are TNF-α-induced nuclear extracts (0.55 mg/mL) + specific probes (1µM) (red column), BSA (1 mg/mL) + specific probes (1µM) (green column), and TNF-α-induced nuclear extracts (0.55 mg/mL) + nonspecific probes (1µM) (blue column). F and F0 are the fluorescence intensity in the presence and absence of protein, respectively. Error bars show the standard deviation of three experiments.

Inhibition Assay. In resting cells, NF-κB is inhibited by the members of IκB family in the cytoplasm.33 Once activated by ultraviolet irradiation, cytokines, bacterial and viral products, NF-κB is released from IκB.33 The over-activation of NF-κB has been found to be associated with autoimmune and inflammatory diseases, and thus NF-κB is considered as an important target for drug development and cancer therapy.34,35 To demonstrate the feasibility of the proposed method for NF-κB p50 inhibition assay, we used oridonin as the model NF-κB 18


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inhibitor.36 As shown in Figure 5, oridonin induces significant decrease in the value of (F-F0)/F0 (F and F0 are the fluorescence intensity in the presence and absence of nuclear extracts, respectively) (Figure 5, green column) compared with the control without inhibitor (Figure 5, red column), suggesting that oridonin can inhibit the binding of NF-κB p50 to the TF-binding probes.

Figure 5. Measurement of fluorescence intensity in response to 0.25 mg/mL nuclear extracts in the absence (red column) and presence of oridonin (green column). The oridonin concentration is 20 µM. F and F0 are the fluorescence intensity in the presence and absence of nuclear extracts, respectively. Error bars show the standard deviation of three experiments.

CONCLUSIONS In summary, we have developed a simple and sensitive fluorescent method to directly detect TF in crude nuclear extracts using target-actuated isothermal amplification-mediated fluorescence enhancement with fluorescent base 2-AP as the fluorophore. The specific binding of TF with the linear TF-binding probes can initiate the extension reaction in both directions and release ssDNAs. Because of the phosphorothioate modification between two binding sites, the unbound probe is protected from being digested by Exo III and 19



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is partially preserved, which can hybridize with the released ssDNA to initiate cycles of nicking-digestion-hybridization, releasing abundant free 2-AP for enhanced fluorescence signal. Consequently, all the TF-binding probes take part in the amplification reaction no matter they bind with TF or not, greatly improving the detection signal. In contrast to the conventional fluorescent probes for TF assay with the involvement of both dye and quencher,16-19 the 2-AP is quenched through its stacking interaction with the adjacent bases without the involvement of any extra quenchers. In comparison with the reported methods for TF assay,12-15,20-26 this method does not involve any radioisotope labels, separation steps, external primers and templates for the initiation of nucleic acid amplification. Because of the low background signal resulting from efficient stacking interaction of 2-AP with the bases of TF-binding probes, the involvement of both bound and unbound TF-binding probes in the amplification reaction and exonuclease-assisted recycling signal amplification, the proposed method can sensitively detect NF-κB p50 in nuclear extracts with a detection limit of as low as 4.11 × 10-4 mg/mL, which is superior to the reported methods for TF assay (Table 2). Importantly, this method can be applied for the screening of potential TF inhibitors, and it may be extended to selectively detect a variety of DNA-binding proteins by simply changing the binding-site sequences of the probes.

AUTHOR INFORMATION Corresponding Author *Tel.: +86 0531-86186033. Fax: +86 0531-82615258. E-mail: [email protected]. *Tel.: +86 0531-86180010. Fax: +86 0531-86180017. E-mail: [email protected]. 20


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Author Contributions †These authors contributed equally. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (Grant Nos. 21325523, 21527811, 21735003 and 21605098), and the Award for Team Leader Program of Taishan Scholars of Shandong Province, China.

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For TOC only

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Sensitive Detection of Transcription Factor in Nuclear Extracts by

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