Nanopore-Based DNA-Probe Sequence-Evolution Method Unveiling

Mar 9, 2015 - Key Laboratory for Large-Format Battery Materials and Systems, Ministry of Education School of Chemistry and Chemical Engineering, Huazh...
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Nanopore-Based DNA-Probe Sequence-Evolution Method Unveiling Characteristics of Protein−DNA Binding Phenomena in a Nanoscale Confined Space Nannan Liu,§,⊥ Zekun Yang,§,⊥ Xiaoding Lou,§ Benmei Wei,§ Juntao Zhang,§ Pengcheng Gao,§ Ruizuo Hou,§ and Fan Xia*,§,‡ §

Key Laboratory for Large-Format Battery Materials and Systems, Ministry of Education School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology (HUST), Wuhan, Hubei 430074, P. R. China ‡ National Engineering Research Center for Nanomedicine, Huazhong University of Science and Technology (HUST), Wuhan, Hubei 430074, P. R. China S Supporting Information *

ABSTRACT: Almost all of the important functions of DNA are realized by proteins which interact with specific DNA, which actually happens in a limited space. However, most of the studies about the protein−DNA binding are in an unconfined space. Here, we propose a new method, nanopore-based DNA-probe sequenceevolution (NDPSE), which includes up to 6 different DNA-probe systems successively designed in a nanoscale confined space which unveil the more realistic characteristics of protein−DNA binding phenomena. There are several features; for example, first, the edge-hindrance and core-hindrance contribute differently for the binding events, and second, there is an equilibrium between protein−DNA binding and DNA−DNA hybridization.

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environment, not like the realistic above phenomena happening in a very “crowded” space, such as in the micro/nanoscale nucleus/nucleosome.14 Research of site-specific DNA binding proteins finding their targets in a nanoscale confined space, therefore, may unveil the more realistic characteristics of protein−DNA binding phenomena. In the meantime, many nanoscale confined spaces in real living systems play very important roles, for example, the ion channels, embedded within cell membranes, communicating chemically and electrically with the extracellular world.15 There is much enthusiasm in the scientific community to study the artificial nanopores,16,17 which supply a suitable platform for the mimic of cellar membrane,18−20 DNA sequencing,21 molecular filtration,22 biomolecular detection,23−25 and nanofluidic devices.26−28 In order to study protein−DNA binding phenomena in a more realistic spacial environment, here, we propose a new method named nanopore-based DNA-probe sequence-evolution (NDPSE) as shown in Figure 1, which includes 6 different DNA-probe systems successively designed in a nanoscale confined space. We choose DNA binding activity of TATA binding protein (TBP),29,30 as a model. NDPSE studies both the DNA-probe system with a single TBP binding site and the

lmost all of the biological/chemical functions of nucleic acid are realized by certain proteins that interact with specific nucleic acid sequences,1,2 and the conversant example occurs at the starting point of DNA replication, during the period of gene expression by RNA polymerase.3 Many studies focus on different aspects, for example: (1) How site-specific DNA binding proteins find their targets, which show how nonspecific DNA−protein interactions may account for accelerated targeting.4 (2) Researchers, who demonstrate protein−DNA binding complexities and multiprotein codes, summarize findings from structural and biochemical studies which reveal the reason why there are no simple protein−DNA recognition codes.5 (3) Traditional approaches for protein− DNA binding detection contain gel mobility shift assays,6 DNA footprinting,7 and enzyme-linked immunosorbent assays (ELISAs)8 which tend to be tedious and time-consuming. Nowadays, the fluorescence spectrum,9 molecular beacons,10,11 and nucleic acid aptamer technologies12 offer the potential for rapid, sensitive, and specific detection which are compatible to high-throughput quantitative analysis. One important thing, however, seems to be neglected. The motions and the bindings between a certain protein (for instance, transcription factors) and its specific DNA strand are completely dominated by the surrounding other proteins and DNA strands in a very limited space (for example, nanoscale space).13 However, almost all of the studies about protein− DNA binding phenomena occur in an unlimited spatial © 2015 American Chemical Society

Received: January 29, 2015 Accepted: March 9, 2015 Published: March 9, 2015 4037

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DNA-probe system with two TBP binding sites, which unveil the valid dominant binding site when there is more than one binding site in the nanoscale confined space, and the protein could not bind with its specific DNA strand due to the steric hindrance. Moreover, the interactions between the above two kinds of probes and TBP reveal the different stability among various DNA-probe systems and diverse DNA−TBP complexes. Finally, we choose one of the DNA-probe systems and accomplish a quantitative detection of DNA binding activity with ultrasensitivity and specificity, providing significant advantages over existing methods for the detection of DNA binding activity.

Sinopharm Chemical Regent (Beijing, China). All DNA sequences are synthesized and purified by TaKaRaba Biotech (Dalian, China). The DNA sequences are listed in Table S1, Supporting Information. TATA boxing protein is purchased from Abnova (Taiwan, China). Streptavidin, catalase, and bovine serum albumin are purchased from Aladdin. All chemical reagents are used as received without further purification. The water used in this project is ultrapure water. Fabrication of the Nanopores. Poly(ethylene terephthalate) films (PET, approximately 12 μm thick) are irradiated with an Au ion beam (11.4 MeV/u at GSI, Darmstadt). The track density is approximately ∼1 × 108/cm2. Cylindrical nanochannels are fabricated through a previously published method.2 Briefly, PET films are pretreated with UV irradiation (approximately 1.0 mW/cm2) for 1 h on each side before etching with NaOH (2 M) at 50 °C for 5 min. After that, the films are thoroughly washed with and restored in ultrapure water. The morphology of the nanochannels is imaged with field emission scanning electron microscopy (Nova Nano SEM 450) (Figure S1, Supporting Information). For the system, the pore diameter is 34 ± 3 nm, with over 50 measurements. Chemical Modification of Nanopores. The DNA Pab is immobilized onto the PET surface and the inner pore wall by a two-step chemical reaction (Figure S2, Supporting Information).3 The NHSS ester is formed by exposure of the PET film to an aqueous solution of 15 mg of EDC and 3 mg of NHSS for 8 h. These PET-NHSS ester monolayers are reacted for 10 h with a solution of 5′-aminated DNA (the probe Pab, 1 μM) in 600 μL of ultrapure water. After reaction with DNA, some of the NHSS byproduct is removed by distilled water. The reactions are taken place at 4 °C. Measurement of the Transmembrane Ionic Current. The self-made two-compartment electrochemical cell is constructed on the basis of the device which is described in our previous work.3 There are two apertures on the top of each cell: one is for the electrode, and the other is used for the thermometer. Ag/AgCl (1 mm in diameter and 20 mm in length) acts as the electrode for current measurements in electrolyte, 50 mM sodium phosphate and 1500 mM NaCl, pH 7.4. The pH of the electrolytes is adjusted by 0.1 M HCl and 0.1 M NaOH. The transmembrane ionic currents are all measured by a Keithley 6487 picoammeter which can provide the scanning voltage from −2 to 2 V. The effective area for the measurement is about 7 mm2. After chemical modification of nanopores with DNA probe Pab, different concentrations of TBP solution, ranging from 1 pM to 10 nM, were used to merge chemical modified PET films with proper buffer solution (50 mM sodium phosphate, 150 mM NaCl, pH 7.4) for 10 h. After the reaction with TBP, some of the residues were removed by ultrapure water. The reaction of DNA probe Pab and any other DNA probes proceeds in the same manner as the reaction of TBP in different buffer solutions of a 10 mM Tris solution (pH = 7.4, 500 mM NaCl, 1 mM MgCl2).

EXPERIMENTAL SECTION Materials and DNA Sequences. 1-Ethy-3-(3(dimethylamino)propyl) carbodiimide hydrochloride (EDC· HCl), N-hydroxysuccinimide (NHSS), and streptavidin are purchased from Sigma-Aldrich. Tris(hydroxymethlyl) aminomethane (Tris) is purchased from Alfa Aesar. Magnesium chloride hexahydrate (MgCl2·6H2O, 98%), sodium chloride (AR), and sodium phosphate (AR) are purchased from

RESULTS AND DISCUSSION In NDPSE, protein−DNA binding phenomena are carried out in a nanoscale confined space. Two kinds of steric hindrances should be considered: one from the edge (just like the part near the membrane of the nucleus) and the other from the core (just like the core of the nucleus full of chromosomes and many other proteins). DNA-probes with a single binding site (TATA box, a DNA sequence found in the promoter region of genes in

Figure 1. Totally, 6 different DNA-probes are introduced into a cylindrical nanopore array. After adding TBP into the buffer, the change of transmembrane ionic current conductance is studied in NDPSE, respectively (A−F). The distinctive conductance reductions are found, which means that the proteins find their specific binding sites, except the proteins in (B).

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4038

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Analytical Chemistry archaea and eukaryotes) are first modified, via a two-step chemical reaction, into a cylindrical nanopore array in a poly(ethylene terephthalate) membrane which is prepared first through UV treatment and then chemical etching in a 2 M NaOH solution at 50 °C (Figures S1 and S2, Supporting Information). After adding TBP into the buffer, they diffuse from the external solution into the nanopore, following targeting at the TATA box in the DNA-probe. The binding between TBP and DNA-probe decreases the effect diameter of the nanopore; that is, it switches off the transverse ionic flux, leading to a highly efficient nanofluidic gating system. Finally, the transmembrane ionic current reduces by 87.5% (Figure 1A). All of the binding between DNA-probe and TBP occurs in 10 h, and the incubation buffer for the bindings contains 50 mM sodium phosphate and 150 mM NaCl, pH 7.4; all of the ionic conductance is measured in electrolyte (50 nM sodium phosphate, 1500 nM NaCl, pH 7.4) (Figure S3, Supporting Information). 1500 mM NaCl is used to stabilize the ionic conductance measurement, and the measurement process is less than 1 min. The DNA-probe, Pab, has two confirmations with a predictable switching free energy ΔΔG of −0.54 kcal/ mol, favoring the binding state (For the architecture presented here, we have used mfold to calculate the free energy, and the mfold web server is tremendously used in computational biology; http://mfold.rna.albany.edu/). Upon the TBP binding, the conformation (ΔG = −5.57 kcal/mol) without the binding site switches into the conformation (ΔG = −6.11 kcal/ mol) with the only binding site (Figure S4, Supporting Information), demonstrating that the TBP-TATA box binding occurs in the nanoscale confined space even under the edge and core-steric hindrances. After interactions of the TBP to DNA, the change of surface charges will also affect the ionic current31−36 besides the size effect. Our experiments show that the signal decrease for the binding between TBP and DNA-probe is saturated for the 10 h incubation, which reveals the equilibrium between the change of surface charges (impedes infinite TBP binding with the DNA-probe) and the size effect (induces the signal decrease). The duplex-DNA-probe, PabPa* (a and a* could hybridize to form a TATA box, near the edge of the nanopore, in Figure 1B), with two DNA strands but also with only one binding site, is introduced here. Interestingly, after adding TBP into the buffer, the transmembrane ionic current is kept almost the same, which shows that the TBP does not bind with the designed PabPa* (Figure 1B). Since the Pab has chemical binding with the nanopore, not Pa*, the above results also illustrate that the duplex-DNA-probe, PabPa* complex, does not dissociate even upon TBP. Otherwise, the remainder Pab in the nanopore should have been binding with TBP, inducing the decrease of transmembrane ionic current. TBP not binding with PabPa* (Figure 1B) reveals that the edge steric hindrance dominants the process here. Another duplex-DNA-probe, PabPb* (b and b* could hybridize to form a TATA box, near the core of the nanopore, in Figure 1C), consisting of two DNA strands but still with only one binding site, is introduced here. In contrast to the behavior of PabPa*, after adding TBP into the buffer, the transmembrane ionic current reduces by 69.6% (Figure 1C), contributed by two possible binding events (TBP with PabPb* duplex or TBP with Pab dissociated single strand from the PabPb*). Because of (1) the PabPa* duplex not dissociating upon TBP introduction (Figure 1B) and (2) the same free energy for PabPa* (ΔG = −40.41 kcal/mol) and PabPb* (ΔG = −40.43 kcal/mol), the

PabPb* duplex does not dissociate upon adding TBP either (Figure 1C). The binding between TBP and the PabPb* duplex, thus, mainly accounts for the reduction of the transmembrane ionic current. Therefore, Figure 1B,C shows that the binding between TBP and DNA-probe with a single-binding site could overcome the core-steric hindrance but not the edge-steric hindrance. In order to further confirm the above the conclusion, we design the third duplex-DNA-probe, Pab ′ Pb* ′ (b and b* could hybridize to form a TATA box, near the core of the nanopore, in Figure 1D), consisting of two DNA strands but also with only one binding site. Neither P′ab nor P′b* could bind with TBP; therefore, the binding between TBP and Pab ′ Pb* ′, with the same length as PabPb* (47-mer), accounts for the reduction of the transmembrane ionic current (Figure 1D), which further illustrates the core-steric hindrance improbably impeding the DNA−protein binding in a nanoscale confined space. The fourth duplex-DNA-probe, PabPa*b* (a and a* and b and b* could hybridize to form TATA boxes, near the edge and the core of the nanopore), has two binding sites (Figure 1E). PabPa*b*, itself, has two binding sites, together with Pab’s ability of binding with TBP; thus, there are totally three possible opportunities for the reduction of ion current in Figure 1E. In consideration of the free energy of PabPa*b* (ΔG = −48.35 kcal/ mol much higher than that of PabPa* and PabPb*) (Figure S4, Supporting Information), together with the conclusions from Figure 1B−D, we could deduce that the upper binding site (b and b*), not the bottom one (a and a*), is the dominant one for the binding phenomena between TBP and DNA with a TATA box. The last duplex-DNA-probe, PabP (ΔG = −32.49 kcal/mol), without a binding site, surprisingly “could” bind with TBP in the nanopore (Figure 1F). Moreover, the “Signal Decrease Percentage” in Figure 1F is higher than that in Figure 1C−E (Figure S5, Supporting Information). Thus, only the dissociation between Pab and P in the duplex probe, following the left Pab binding with TBP, could explain the above “strange phenomena”. We, therefore, gain another useful conclusion that the binding between protein and DNA could replace the hybridization between DNA and its complementary strand with relatively low free energy. Until now, NDPSE illustrated that both the DNA-probe system with a single binding site (1 type) and the DNA-probe system with two binding sites (5 types) could bind with TBP, which unveils the valid dominant binding site when there are more than one in the nanoscale confined space. We also are curious about the interactions between the above two kinds of probes and TBP, which may reveal the different stability among various DNA-probe systems and diverse DNA−TBP complexes. The single strand DNA probe Pab is the beginning state, followed by adding Pa*b*, and they form the PabPa*b* complex (Figure S6, Supporting Information). The ionic current increase is due to the increasing hydrophilicity (dsDNA is more hydrophilic than ssDNA).37 Adding TBP into the buffer, in the following step, will decrease the ion current (Figure 2A) due to the similar reasons stated in Figure 1E. In Figure 2B, we change the order of addition, first TBP and then Pa*b*. Interestingly, the current first decreases due to the similar reason in Figure 1A and then increases, which means the hybridization between Pab and Pa*b* could partially replace the binding between Pab and TBP. In addition, Figure 2C actually explains why the “strange” phenomena happen in Figure 1F in detail. However, Figure 2D proves that the hybridization between Pab and P, with relatively low free 4039

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Figure 3. Reagentless, Pab, drastically simplifies the detection process. The calibration curve for the detection of TBP reveals a very sensitive detection ability 1 pM (A). Different control experiment data are listed in (B). We, moreover, challenge the detection by adding 1 μM SA to the 10 nM TBP detection process. Pab still could accomplish the sensor’s mission, when adding either TBP first or SA first (C and D).

Figure 2. Single strand DNA probe Pab is the beginning state, followed by adding Pa*b* first and then TBP finally (A); adding TBP first and then Pa*b* finally (B); adding P first and then TBP finally (C); adding TBP first and then P finally (D).

energy, could also partially replace the binding between Pab and TBP, which reveals an equilibrium between the protein−DNA binding and DNA−DNA hybridization. Monitoring fluctuating TF expression levels provides an important assessment of the state of cell populations (e.g., human embryonic stem cell differentiation into neural precursors is often assessed by monitoring the TFs Oct4 and Sox140). However, traditional detection methods bear different drawbacks.38 After carefully studying the TBP with different DNA-probes through NDPSE and comparison of different signal changes in Figure 1, we choose Pab as the detection probe for TBP, which drastically simplifies the detection of active DNA binding proteins by eliminating washing and/or transfer steps (e.g., ELISA, Western blots) and worries about the annoying bleach effects (e.g., fluorescence-related methods).39−42 Figure 3A exhibits the calibration curve for the detection of TBP, with a very sensitive detection ability of 1 pM (Table S2, Supporting Information). Figure 3B demonstrates the specificity for the detection (both the concentration of TBP and competing proteins are 10 nM). We, moreover, challenge the detection by adding 1 μM SA to the 10 nM TBP detection process (Figure 3C). Pab still could accomplish the sensor’s mission, when adding either TBP first or SA first (Figure 3D). Therefore, the Pab evolution from NDPSE not only acts as a quantitative detection of DNA binding activity with ultrasensitivity and specificity, providing significant advantages over existing methods, but also is likely generalizable to the detection of other TFs.

addition, the equilibrium between protein−DNA binding and DNA−DNA hybridization events depends on their own free energy. Finally, Pab evolution from NDPSE does not only act as an ultrasensitive and specific sensor for TBP detection but also is likely generalizable to the detection of other TFs.

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ASSOCIATED CONTENT

S Supporting Information *

Detailed description of the experimental procedures, DNA sequences, and additional figures. 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]. Author Contributions ⊥

N.L. and Z.Y. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research is supported by National Basic Research Program of China (973 program, 2015CB932600, 2013CB933000), National Natural Science Foundation of China (21375042, 21405054), and 1000 Young Talent (F.X.).

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CONCLUSION In brief, we develop a new method named nanopore-based DNA-probe sequence-evolution (NDPSE), by successively designing different DNA-probes, which reveals several characteristics of protein−DNA binding phenomena in a nanoscale confined space. First, the sequence dependent protein could bind with both a single-binding site DNA and multibinding-site DNA. Second, when there is more than one binding-site, one of them is the dominant one. Third, some binding sites could not bind with target protein due to the steric hindrance. In 4040

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