Real-Time Detection of Transcription Factors Using Target-Converted

Jan 15, 2013 - Real-Time Detection of Transcription Factors Using Target-Converted Helicase-Dependent Amplification Assay with Zero-Background Signal...
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Real-Time Detection of Transcription Factors Using TargetConverted Helicase-Dependent Amplification Assay with ZeroBackground Signal Anping Cao and Chun-yang Zhang* Single-Molecule Detection and Imaging Laboratory, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Guangdong 518055, China ABSTRACT: Highly sensitive detection of transcription factors is essential to the evaluation of cellular development and the disease state. However, so far most detection methods are usually laborious and time-consuming with a poor sensitivity. Here, we demonstrate a simple and ultrasensitive approach for transcription factor detection based on the target-converted helicase-dependent amplification assay. We employ a hairpin probe bearing the transcription factor binding site to convert the protein signal to the DNA signal, which can be further amplified by helicasedependent amplification. With the digestion of excess probes by the exonucleases and the subsequent one primer-triggered high fidelity amplification, zerobackground signal can be achieved in the absence of a transcription factor. This method exhibits excellent specificity and high sensitivity with the detection limit of 9.3× 10−13 M and the detection range over 4 orders of magnitude, which is superior to most currently used approaches for transcription factor detection. Moreover, the proposed method has significant advantages of simple, rapid, and low cost without the need of any labeled DNA probes and might be extended to selectively detect various DNA-binding proteins by simply changing the binding-site sequences of hairpin probes.

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containing half of the protein-binding site are labeled with a fluorescence donor and an acceptor, respectively. In the presence of transcription factors, the formation of DNA duplexes results in a high FRET signal.13 Due to the short dynamic range of FRET (1−10 nm), this assay requires close proximity between the donor and the acceptor, but the protein−DNA binding might induce steric hindrance, leading to a low FRET signal.2 Therefore, the development of new methods for sensitive and selective detection of transcription factors is highly desirable. In order to improve the detection sensitivity, two strategies of signal amplification and background reduction are usually employed. The signal amplification strategy includes polymerase chain reaction (PCR) and a variety of isothermal DNA amplification methods such as strand displacement amplification (SDA),14 exponential amplification reaction (EXPAR),15 and loop-mediated isothermal amplification (LAMP).16 These methods rely on the template replication and might suffer from the cross contamination of amplicons and the false positivity.17 Alternatively, helicase-dependent amplification utilizes a helicase to separate dsDNA without the need for strand displacement activity of DNA polymerase.18−20 The background reduction strategy usually employs the nanoparticles such as gold nanoparticles, carbon nanotubes, and graphene

ranscription factors are sequence specific DNA-binding proteins that modulate the process of gene transcription by binding to a specific double-stranded DNA (dsDNA) sequence,1 and they may function as natural switches to translate physical and chemical signals such as temperature shifts, light exposure, chemical concentrations, and redox status into transcriptional changes by modulating the binding of RNA polymerase to promoter DNA.2 Transcription factors play pivotal roles in the pathways and networks of gene expression regulation, and their expression levels sensitively reflect cellular development and disease state. Consequently, they have become potential targets in medical diagnosis and drug development.3 Recently, nuclear factor-kappa B (NF-κB),4 a ubiquitous transcription factor that is involved in the regulation of a large number of genes and closely related to some diseases, has already become an important target for drug development.5 Therefore, the methods for sensitive detection of transcription factors might provide both fundamental information about gene regulation and a platform for drug development. Although a variety of methods have been developed for transcription factor detection, such as electrophoretic mobility shift assay (EMSA),6 DNA footprinting,7 enzyme-linked immunosorbent assay (ELISA),8 and Western blotting assay,9 these methods are usually laborious and time-consuming with poor sensitivity. Alternatively, fluorescence-based methods have been employed for the detection of transcription factors in a homogeneous way.10−12 In a typical fluorescence resonance energy transfer (FRET) assay,13 two short DNA duplexes © 2013 American Chemical Society

Received: January 1, 2013 Accepted: January 15, 2013 Published: January 15, 2013 2543

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B. Mix A (25 μL) contained 20 μL of digestion mixture, 1 μL of 100 mM MgSO4, 2 μL of 500 mM NaCl, and 1 μL of 5 μM primer. Mix A was incubated at 95 °C for 5 min and then slowly cooled to 65 °C over 5 min. Mix B (25 μL) contained 5 μL of 10× annealing buffer II, 1 μL of 100 mM MgSO4, 2 μL of 500 mM NaCl, 3.5 μL of IsoAmp dNTP solution, and 0.05× SYBR Green I. The helicase-dependent amplification reaction was performed at 65 °C in a total volume of 50 μL solution containing 25 μL of mix A and 25 μL of mix B. The real-time fluorescence measurements were performed in a Roche Light Cycler nano (Switzerland), and the fluorescence intensity was monitored at intervals of 40 s.

oxide as the quenchers to reduce the background signal21−24 but few can achieve the zero-background signal. Notably, so far there are few reports about the detection methods which combine the advantages of both signal amplification and zerobackground signal.25 In this research, we develop for the first time a target-converted helicase-dependent amplification assay which combines both signal amplification and zero-background signal for sensitive detection of transcription factors. The DNA binding protein NF-κB p50 is a member of a family of eukaryotic transcription factors, synthesized from a 105 kDa precursor protein through proteolytic cleavage to yield the mature transcription factor.26 NF-κB p50 belongs to the category of “rapid-acting” primary transcription factors and plays an important role in regulating the cellular responses to harmful cellular stimuli. In this research, NF-κB p50 is selected as the model protein. We employed a hairpin probe bearing the transcription factor binding site to convert the protein signal to the DNA signal, which can be further amplified by helicasedependent amplification. With the digestion of excess probes by the exonucleases and the subsequent one primer-triggered high fidelity amplification, a zero-background signal can be achieved in the absence of a transcription factor.

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RESULTS AND DISCUSSION Principle of Target-Converted Helicase-Dependent Amplification Assay for Real-Time Detection of Transcription Factors. The principle of transcription factor detection is illustrated in Scheme 1. The hairpin probe consists Scheme 1. Principle of Target-Converted HelicaseDependent Amplification Assay for Transcription Factor Detection

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EXPERIMENTAL SECTION Materials. The hairpin probe (5′-AGT ATG GGA CTT TCC GTC TCA TCA CAA CTA CAG AGA CAG ATA CTT AAT ACT AGT TGT GAT GAG ACG GAA AGT CCC ATA CT-3′, the underlined regions indicate the binding site of NFκB p50), the primer (5′-AGT ATG GGA CTT TCC GTC TCA TCA CAA CTA-3′), the probe for the control experiment (5′-AGT ATA GAC ATG CCT AGA CAT GCC TAA CTA CAG AGA CAG ATA CTT AAT ACT AGT TAG GCA TGT CTA GGC ATG TCT ATA CT-3′), the primer for the control experiment (5′-AGT ATA GAC ATG CCT AGA CAT GCC TAA CTA-3′), and the deoxynucleotide mixture (dNTPs) were purchased from Takara Biotechnology Company Ltd. (Dalian, China). Before use, 5 μM hairpin probes were incubated in a buffer containing 1.5 mM MgCl2 and 10 mM Tris-HCl (pH 8.0) at 95 °C for 5 min and then slowly cooled to room temperature over 30 min to make the probes perfectly folded into hairpin structures. The purified recombinant NF-κB p50 (rhNF-κB p50) was purchased from Promega (Madison, WI). Exonuclease III (Exo III) and Exonuclease I (Exo I) were obtained from New England Biolabs (Ipswich, MA, USA). IsoAmp II Universal tHDA kits were obtained from BioHelix Corp. (Beverly, MA, USA), and it contained IsoAmp enzyme mix, IsoAmp dNTP solution, 10× annealing buffer II, 100 mM MgSO4, 500 mM NaCl, 1 ng/μL pCNG1, 5 μM NGF3, and 5 μM NGR3. SYBR Green I was purchased from Xiamen BioVision Biotechnology (Xiamen, China). Protein−DNA Interaction and Exonuclease Digestion. The purified recombinant NF-κB p50 and hairpin probes were incubated at 37 °C for 20 min in 10 μL of 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, 1 μL of 1 μM hairpin probe and a different concentration of NF-κB p50. Then, 50 units of Exo III, 20 units of Exo I, and 2 μL of 1× NEBuffer 1 were added to digest the hairpin probes at 37 °C for 20 min. The digestion reaction was terminated by heating at 95 °C for 10 min. Helicase-Dependent Amplification Reaction and RealTime Detection. The helicase-dependent amplification reaction mixtures were prepared separately as mix A and mix

of two regions: a stem region containing the protein-binding site of the transcription factor and a loop region containing the additional nucleotide sequences. In the absence of transcription factors, the hairpin probes are completely digested by Exo III and Exo I and no hairpin probe remains, thus a zerobackground signal is observed. However, in the presence of transcription factors which can specifically bind to the proteinbinding site in the stem region of hairpin probes, the digestion is blocked, making the hairpin probes retain the stem-loop structure. With the addition of polymerase and primer which is complementary to the stem oligonucleotides, the remaining hairpin probes will open to yield dsDNAs as a result of primer extension. The resultant dsDNAs can serve as the templates for helicase-dependent amplification, generating large numbers of amplified products for the next round of amplification. The amplified dsDNA products can simply be detected by SYBR Green I. It should be noted that only one kind of primer is able to trigger the exponential amplification in this assay. Detection of NF-κB p50 with Target-Converted Helicase-Dependent Amplification Assay. Since SYBR Green I exhibits negligible fluorescence in the presence of short single-stranded DNA (ssDNA),27 but distinct flurescence signal in the presence of dsDNA, SYBR Green I is used as the 2544

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fluorescent dye for label-free, real-time detection of NF-κB p50. In the presence of NF-κB p50, the binding of NF-κB p50 with the probes can protect the hairpin probes from digestion by Exo III and Exo I, and the protected hairpin probes can transform into dsDNAs in the presence of a primer as a result of primer extension. The resultant dsDNAs can serve as the templates to generate large numbers of dsDNA products through helicase-dependent amplification. As shown in Figure 1, the fluorescence intensity increases in a sigmoidal fashion in

Figure 1. (A) Real-time fluorescence monitoring of helicasedependent amplification in the presence of 100 nM NF-κB p50 (red line) and in the absence of NF-κB p50 (black line). (B) Electrophoretic identification of the products obtained from helicasedependent amplification by 2% agarose gel electrophoresis. Lanes 1 and 2 represent the amplification products in the presence of 100 nM NF-κB p50 and in the absence of NF-κB p50, respectively. Lane 3 represents the hairpin probes only. Figure 2. Sensitive detection of NF-κB p50. (A) Real-time fluorescence curves obtained from the amplification triggered by different concentration NF-κB p50. (B) Linear relationship between the fluorescence intensity and the logarithm of NF-κB p50 concentration. The fluorescence intensities in Figure 2B are obtained at 30 min. Error bars show the standard deviation of three experiments.

the presence of 100 nM NF-κB p50 (Figure 1A, red line). While in the control group without NF-κB p50, the hairpin probes are completely digested by Exo III and Exo I, thus no helicase-dependent amplification occurs and zero-background signal is observed (Figure 1A, black line). The observed zerobackground signal might be attributed to the following two factors: (1) the hairpin probes are completely digested by Exo III and Exo I in the absence of NF-κB p50, and the helicasedependent amplification cannot be initiated without the hairpin probes. (2) Only one kind of primer is used in the helicasedependent amplification, thus eliminating the “primer−dimer”type nonspecific background amplification observed in the traditional amplifications.28 To further verify this assay, the products obtained from helicase-dependent amplification are analyzed by 2% polyacrylamide gel electrophoresis (Figure 1B). A well-defined band of amplified products is observed in the presence of 100 nM NF-κB p50 (Figure 1B, lane 1), which moves much slower than the hairpin probes (Figure 1B, lane 3); but, no band is observed in the control group without NF-κB p50 (Figure 1B, lane 2) because the hairpin probes are digested completely by the exonucleases in the absence of protection from NF-κB p50. These results are consistent with those of real-time fluorescence measurement (Figure 1A), suggesting the target-converted helicase-dependent amplification assay can sensitively detect translation factors with excellent specificity. Improved Sensitivity for NF-κB p50 Detection. To demonstrate the improved sensitivity of the proposed assay, we detect NF-κB p50 at various concentrations by real-time fluorescence measurement. As shown in Figure 2A, the fluorescence intensity increases monotonically with the increase of NF-κB p50 concentration, suggesting that the concentration

of NF-κB p50 is directly proportional to the products of hairpin probe-specific helicase-dependent amplification. Moreover, in logarithmic scales the fluorescence intensity exhibits a linear correlation with the concentration of NF-κB p50 through a detection range over 4 orders of magnitude from 1 pM to 10 nM (Figure 2B). The regression equation is F = 0.08926 + 0.00742 log10 C with a correlation coefficient of 0.990, where F and C represent the fluorescence intensity and the NF-κB p50 concentration (molar), respectively. The detection limit is calculated to be 9.3 × 10−13 M. Notably, the sensitivity of the proposed assay has improved by as much as 4 orders of magnitude as compared with the previously reported FRETbased assay (5 nM and 20 nM, respectively)10,13 and the labelfree luminescence assay (30 nM)29 and up to 4 orders of magnitude as compared with AuNP-based colorimetric assay (10 nM) as well.30 Such significant improvement in the detection sensitivity might be attributed to the combination of hairpin probe-specific helicase-dependent amplification with the zero-background signal. Detection Specificity. To verify its specificity, the proposed assay was challenged with two control experiments that involved (1) a nonspecific sequence of NF-κB p50 and (2) an irrelevant protein of bovine serum albumin (BSA), respectively. In the first experiment, we encoded the NF-κB p50-binding sequences into the stem region of the hairpin 2545

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been used as an important drug target.32,33 In this research, oridonin was selected as a model inhibitor34 to demonstrate the feasibility of the proposed assay for screening inhibitors of NFκB. Figure 4A displays the real-time fluorescence in response to

probe. Equal concentrations of specific and nonspecific sequence of NF-κB p50 were incubated with NF-κB p50, respectively. After digestion and amplification, the real-time fluorescence intensity increases in a sigmoidal fashion in the presence of a specific sequence (Figure 3A, blue line); however,

Figure 3. (A) Real-time fluorescence curves in response to 20 nM NFκB p50 in the presence of specific probes (blue line), 20 nM NF-κB p50 in the presence of nonspecific probes (red line), and 20 nM BSA in the presence of specific probes (black line), respectively. (B) Detection specificity measured by the fluorescence intensity. The samples tested are NF-κB p50 in the presence of specific probes (p50), BSA in the presence of specific probes (BSA), and NF-κB p50 in the presence of nonspecific probes (control). The fluorescence intensities in Figure 3B are obtained at 30 min. Error bars show the standard deviation of three experiments.

Figure 4. (A) Real-time fluorescence curves in response to 100 nM NF-κB p50 in the absence (red line) and in the presence of 20 μM oridonin (black line). (B) The inhibition effect of oridonin measured by the fluorescence intensity. The samples tested are 100 nM NF-κB p50 in the absence (red column) and in the presence of 20 μM oridonin (black column). The fluorescence intensities in Figure 4B are obtained at 30 min. Error bars show the standard deviation of three experiments.

NF-κB p50 in the presence (Figure 4A, black line) and absence of oridonin (Figure 4A, red line). In the presence of 20 μM oridonin, a significant reduction in the fluorescence intensity is observed as compared with that without oridonin (Figure 4B), suggesting that oridonin can inhibit the binding of NF-κB p50 to the hairpin probes. These results highlight the feasibility of the proposed assay for high-throughput screening inhibitors of NF-κB.

the control group with the nonspecific sequence does not show any observable fluorescence signal (Figure 3A, red line). In the second experiment, the selectivity of the proposed assay was investigated by replacing the NF-κB p50 with an irrelevant protein of BSA, which showed no binding activity to the specific sequence of NF-κB p50. No fluorescence signal is observed in the presence of BSA (Figure 3A, black line). As shown in Figure 3B, the fluorescence intensity in the presence of NF-κB p50 and the specific sequence is much higher than that of the control group with either the nonspecific sequence or an irrelevant protein BSA. These results clearly demonstrate the high specificity of the proposed assay for translation factor detection. The Inhibition Assay. NF-κB is bound to the inhibitory protein IκB in the cytoplasm. With the influence of activators such as ultraviolet irradiation, cytokines, bacterial and viral products, NF-κB can be released from IκB and become activated.31 Previous research demonstrated that the overactivation of NF-κB was associated with a number of autoimmune and inflammatory diseases, and thus NF-κB had

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CONCLUSIONS In summary, we have developed a simple and ultrasensitive method for transcription factor detection based on targetconverted helicase-dependent amplification assay. This method employs a hairpin probe containing a translation factor-binding sequence to convert the protein signal to the DNA signal, which can be further amplified through helicase-dependent amplification to produce a distinct fluorescence signal. With the digestion of excess probes by the exonucleases and the subsequent one primer-triggered high fidelity amplification, zero-background signal can be achieved in the absence of 2546

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(24) Wang, Y.; Li, Z.; Hu, D.; Lin, C. T.; Li, J.; Lin, Y. J. Am. Chem. Soc. 2010, 132, 9274−9276. (25) Lu, L. M.; Zhang, X. B.; Kong, R. M.; Yang, B.; Tan, W. J. Am. Chem. Soc. 2011, 133, 11686−11691. (26) Ghosh, S.; Gifford, A. M.; Riviere, L. R.; Tempst, P.; Nolan, G. P.; Baltimore, D. Cell 1990, 62, 1019−1029. (27) He, J. L.; Wu, Z. S.; Zhou, H.; Wang, H. Q.; Jiang, J. H.; Shen, G. L.; Yu, R. Q. Anal. Chem. 2010, 82, 1358−1364. (28) Tan, E.; Erwin, B.; Dames, S.; Ferguson, T.; Buechel, M.; Irvine, B.; Voelkerding, K.; Niemz, A. Biochemistry 2008, 47, 9987−9999. (29) Ma, D. L.; Xu, T.; Chan, D. S.; Man, B. Y.; Fong, W. F.; Leung, C. H. Nucleic Acids Res. 2011, 39, e67. (30) Ou, L. J.; Jin, P. Y.; Chu, X.; Jiang, J. H.; Yu, R. Q. Anal. Chem. 2010, 82, 6015−6024. (31) Chen, F.; Castranova, V.; Shi, X.; Demers, L. M. Clin. Chem. 1999, 45, 7−17. (32) Fujisawa, K.; Aono, H.; Hasunuma, T.; Yamamoto, K.; Mita, S.; Nishioka, K. Arthritis Rheum. 1996, 39, 197−203. (33) Aggarwal, B. B. Nat. Rev. Immunol. 2003, 3, 745−756. (34) Leung, C. H.; Grill, S. P.; Lam, W.; Han, Q. B.; Sun, H. D.; Cheng, Y. C. Mol. Pharmacol. 2005, 68, 286−297.

transcription factors. This assay exhibits high sensitivity with a detection limit of 9.3 × 10−13 M and a detection range of 4 orders of magnitude, which is superior to most currently used methods for transcription factor detection.10,13,29,30 Importantly, this method might be further extended to selectively detect various dsDNA-binding proteins by simply changing the binding-site sequences of hairpin probes.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +86 755 86392211. Fax: +86 755 86392299. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Basic Research Program 973 (Grants 2011CB933600 and 2010CB732600), the Award for the Hundred Talent Program of the Chinese Academy of Science, the National Natural Science Foundation of China (Grant 21075129), the Guangdong Innovation Research Team Fund for Low-cost Healthcare Technologies, the Natural Science Foundation of Shenzhen City (Grant JC201005270327A), and the Fund for Shenzhen Engineering Laboratory of Single-molecule Detection and Instrument Development [Grant (2012) 433].

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