Quantifying the Interactions between PEI and Double-Stranded DNA

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Quantifying the Interactions between PEI and Double-Stranded DNA: Toward the Understanding of the Role of PEI in Gene Delivery Xiaolong Kou, Wei Zhang, and Wenke Zhang*

ACS Appl. Mater. Interfaces 2016.8:21055-21062. Downloaded from pubs.acs.org by KAOHSIUNG MEDICAL UNIV on 10/11/18. For personal use only.

State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012, People’s Republic of China ABSTRACT: Poly(ethylene imine) (PEI) is one of the most efficient nonviral vectors, and its binding mode/strength with double-stranded DNA (dsDNA), which is still not clear, is a core area of transfection studies. In this work we used the atomic force microscopy (AFM)-based single molecule force spectroscopy (SMFS) to detect the interaction between branched PEI and dsDNA quantitatively by using a long chain DNA as a probe. Our results indicate that PEI binds to phosphoric acid skeletons of dsDNA mainly via electrostatic interactions, no obvious groove-binding or intercalation has happened. The interaction strength is about 24−25 pN, and it remains unchanged at pH 5.0 and 7.4, which correspond to the pH values in lysosomes and in the cytoplasmic matrix, respectively. However, the interaction is found to be sensitive to the ionic strength of the environment. In addition, the unbinding force shows no obvious loading rate dependence indicative of equilibrium binding/unbinding. KEYWORDS: poly(ethylene imine), AFM, DNA, nonviral vectors, single molecule force spectroscopy

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electrophoresis,16 and so on, by judging the size or morphology change of the complexes. It is quite difficult to derive the information on the stability of PEI/dsDNA complexes and quantitative interactions from those experimental measurements, although theoretical simulations can be used to simulate the interaction.16 The quantification of DNA−PEI interactions as well as the factors that can affect such interactions at the single molecule level will deepen our understanding of the nature of the interaction, and eventually provide the means to tune the interactions and the rational design of new PEI-based gene carriers. AFM-based single molecule force spectroscopy (SMFS) is one of the efficient techniques for the study of molecular interactions at the single molecule level.18−22 Fruitful information has been obtained on the mechanism of DNA melting,23−26 protein unfolding,27−32 nucleic acid protein interactions,33 and so on. In the present study we use the AFM-based SMFS to quantify PEI−DNA interactions under different N/P ratio and pH at the single molecule level. Both the binding mode and binding strength have been acquired directly, and the biological meanings of the study have been discussed.

INTRODUCTION In the most recent three decades, gee therapy was considered to be a promising technique to cure genetic disorders and acquired gene diseases. Due to the presence of serum nucleases in the blood, DNA cannot be injected intravenously to the organisms.1 To overcome this problem, viruses were first used as carriers to send DNA and RNA into the cell.2 However, because of the immunogenicity of virus particle and difficulties of scale-up,3 nonviral vectors then come to be the focus of the research.4,5 Since the 1960s, a variety of synthetic materials have been used as gene carriers. Among them, polycations have become off-theshelf DNA transfection materials.4 Their interaction with DNA have been studied intensively.6,7 Because of the proton sponge effect,8,9 PEI was used as one of the most effective gene-carrier materials since 1995.8,10 Under the physiological pH range, 12−15% of the amino groups of a PEI molecule get protonated.11 As a result, this can interact with DNA through hydrogen bonding12 and electrostatic interactions, which can change the conformation of DNA from a linear chain (or coiled) structure to a compacted globule/nanoparticles structure.13 Many studies have been performed on the mechanism of DNA−PEI interactions, such as molecular dynamics (MD) simulations that showed that PEI forms polycation bridging between negatively charged DNA in the short range.14 With a rise in the N/P ratio (the ratio of nitrogen versus phosphate) and protonated ratio of PEI, the aggregate becomes more compact.15 Electron microscope and electrophoretic mobility shift assay (EMSA) also showed how N/P ratio affects the aggregates.16 However, most of the earlier experimental studies were performed by using ensemble methods, such as imaging,16 dynamic light scattering (DLS),17 © 2016 American Chemical Society

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RESULTS AND DISCUSSION To figure out, at the single molecule level, the binding mode between dsDNA and PEI, one terminal of a dsDNA was attached to the Au substrate by thiol−gold chemistry34 and the other end to the AFM tip by streptavidin−biotin interaction Received: May 29, 2016 Accepted: July 20, 2016 Published: July 20, 2016 21055

DOI: 10.1021/acsami.6b06399 ACS Appl. Mater. Interfaces 2016, 8, 21055−21062

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ACS Applied Materials & Interfaces

Figure 1. Stretching of dsDNA in the presence of PEI. (a) One terminal of dsDNA was attached to the AFM tip, the other end to Au substrate, and dsDNA was stretched in the presence of 0.03 and 0.06 μg mL−1 of PEI. (b) Typical sawtooth force−extension curves. (c) The distribution of rupture length (see b, black arrows) in 0.03 and 0.06 μg/mL PEI, respectively. (d) The dsDNA−PEI aggregates become more compact when more PEI was added. (e, f) The presence of PEI shows no effect on the B-OS transition.

of dsDNA happened at about 65 pN,25 and this transition can be affected differently by small molecules of different binding modes. For example, minor35,36 or major36 groove binders (such

(see Experimental Section). The dsDNA was then stretched in the absence and presence of PEI, as shown in Figure 1a. It was reported before that the overstretching transition (in brief B-OS) 21056


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Figure 2. Histograms of rupture length of dsDNA−PEI complexes with different N/P ratios: (a) 0.75, (b) 1.5, (c) 3.0, and (d) 5.4, in pH 5.0 (left) and pH 7.4 (right).

detectable groove-binding or intercalation to the dsDNA, and suggest that PEI is very likely to bind to the phosphodiester backbone through ionic attraction. There is a long-running debate on the binding mode of the PEI−dsDNA interaction. Different binding modes have been suggested, such as grooving binding,38 bases binding,39 and hydrogen bonding.12 Our data support the phosphodiester backbone binding mode, since any groove-binding effect, if it exists, including the hydrogen-bond-based groove binders like berenil35,40 and electrostatic-interaction-based groove binders like α-helix,36,41 can be clearly identified in the stretching curve.35,36 Obviously, in our system, the 25 kDa branched PEI mainly interacts with the phosphodiester backbone, and neither groove-binding nor intercalation was detectable. It has been shown that the size of PEI−dsDNA complexes depends on the N/P ratio.16 To further study such an effect at the single molecule level, PEI and dsDNA were mixed at different N/P ratios to form the PEI−DNA complexes, which were then immobilized on the gold substrate and stretched by using AFM. To see also the effect of pH on the stability of the PEI−DNA complexes, the stretching experiments were performed in 10% PBS buffer at two different pH values, pH 5.0 and pH 7.4, respectively. Our AFM results show that, at both pHs, with the increase of N/P ratios, the apparent rupture length of DNA molecule was decreased, as shown in Figure 2, in agreement with

as netropsin) can usually increase the plateau force of the B-OS transition.35,37 However, intercalators, like ethidium bromide, can reduce the cooperativity of the B-OS transition, and have manifested as an increasing slope in the transition regime.35,37 Figure 1b shows typical DNA stretching curves in the presence of PEI. A sawtooth pattern can be observed in the force curves. Sometimes the sawtooth pattern was followed by the B-OS plateau. The sawtooth pattern may be attributed to the unraveling of the PEI/DNA complexes since our control experiments demonstrate that these sawtooth peaks do not appear under other conditions such as in pure PEI or dsDNA systems (data not shown). To further prove that, the DNA pulling experiment was performed under PEI concentrations of 0.03 and 0.06 μg mL−1, respectively. Our results show that with the increase of PEI concentration the rupture length (the extension of the last rupture event) of the sawtooth stretching curve gets decreased (from 762 nm under 0.03 μg/mL PEI to 231 nm under 0.06 μg/mL PEI) accordingly, indicating the shortening of the apparent length of dsDNA, as shown in Figure 1c,d. However, our results show that the concentration increase of PEI did not affect the shape of the overstretching force plateau (both the length and flatness) of dsDNA, since the force plateaus appeared at the extension length of >700 nm show good superposition, as shown in Figure 1e,f. These observations show that PEI does not show 21057


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AFM tip to interact with PEI-modified substrate, as shown in Figure 4a (see also Experimental Section and Figure 7). Sawtooth peaks that are similar to those in Figure 1 were observed. Statistical analysis on those small sawtooth peaks appeared before the overstretching force plateau shows the most probable unbinding force of 23.6 pN (at pH 5.0) and 24.9 pN (pH 7.4), respectively, as shown in Figure 4b−d. These two force values are quite similar to those shown in Figure 3, which further proves that the interactions between PEI and dsDNA are around 24−25 pN, and this interaction is not sensitive to pH in the range 5.0−7.4. This phenomenon indicates that PEI/DNA complexes are stable in both the lysosome (∼pH 5.0) and cytoplasm (∼pH 7.2), which lays the foundation for PEI’s gene-carrier function. In addition, we have found that PEI−dsDNA interactions are sensitive to salt concentration. With the increase of PBS concentration from 10% (ionic strength, I = 0.015) to 100% (ionic strength, I = 0.15), the number of sawtooth peaks appeared before the B-OS plateau gets decreased accordingly. In addition, the most probable unbinding force decreased as well. The most probable unbinding force was about 25 pN in 10% PBS (Figure 5b, I = 0.015), 13 pN in 50% PBS (Figure 5d, I = 0.075), and 7 pN in 100% PBS (Figure 5f, I = 0.15). The salt concentration dependence of the unbinding force indicates that the interaction between dsDNA and PEI is dominated by electrostatic interaction under the condition of our SMFS measurement. The unbinding experiments between dsDNA and PEImodified substrate were also performed at different stretching speed, 0.2 μm/s (Figure 6a), 0.5 μm/s (Figure 6b), 1 μm/s (Figure 6c), 5 μm/s (Figure 6d), and 10 μm/s (Figure 6e). Our results show that the unbinding forces were almost the same at different stretching rates, as shown in Figure 6a−f, which suggests that the unbinding process was performed under equilibrium conditions.42−45

the ensemble experiment. As can been seen from Figure 2a, at a lower N/P ratio (e.g., 0.75), some rupture events appear at longer extension (centered at 1300−1500 nm). The dsDNA we used in the current study has 2700 bps; the contour length of such a DNA is around 918 nm (2700 × 0.34). So, the rupture length larger than 918 nm may be attributed to the overstretched dsDNA (during the force-induced B-OS transition, the molecule can reach 1.7 times of its contour length). This is reasonable since, under low N/P ratio (e.g., 0.75), very few PEI molecules will bind to the dsDNA, and some DNA molecules still exist as loose coils. As a result, more bare dsDNA fragments can be stretched before the rupture of the molecular bridge, while with the increase of the N/P ratio, dsDNA can be changed to a compacted globule structure, which causes the shortening of apparent rupture length. In order to quantify the stability of the PEI/DNA complexes, we analyzed the unbinding force of those sawtooth peaks in the stretching curve. To make sure that the single molecule unfolding force is measured, only those sawtooth peaks with the overstretching transition plateau at longer extension, as shown in Figures 1e and 3, were used for statistical analysis. Because the

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CONCLUSIONS In conclusion, by using a long dsDNA as a probe, we studied the binding mode as well as the binding strength between PEI and dsDNA, using AFM-based single molecule force spectroscopy (SMFS). Our results suggest that PEI interacts mainly with the phosphodiester backbone of dsDNA via electrostatic interactions to form aggregates, and no other binding modes (e.g., groovebinding or intercalation) are detectable under our experimental conditions. The interaction strength was about 24−25 pN, and it did not change under pH 7.4 and pH 5.0, which correspond to the pH value in lysosome and cytoplasm, respectively. This fact demonstrates that PEI can bind to dsDNA and protect it from enzymolysis during the gene delivery process.46 In addition, the relatively weak interactions between PEI and dsDNA (from ∼7 to ∼25 pN) under physiological conditions make it possible for a replisome to disassemble the PEI−DNA complexes and use the external DNA as a template to do the replication and expression.47 The established single molecule method may be useful for the study of interactions between other polycations (including their derivatives) and dsDNA.

Figure 3. Force distribution of the sawtooth peaks obtained at (a) pH 5.0, and (b) pH 7.4, respectively. Only those sawtooth force signals that contain the long force plateau of the B-OS transition were selected for the statistical analysis.

peaks are not completely separated, DNA may still interact with PEI even in the low force area, so the statistical analysis was performed on all the data points in the sawtooth peak region (Figure 3a,b, black arrows). Figure 3 shows the force distribution of sawtooth peaks obtained at different pH. From the figure we can see that the most probable unbinding forces at these two pHs are quite close, which is around 25 pN. This result indicates that the stabilities of the PEI/DNA complexes are very similar at pH 5.0 and pH 7.4. To further prove that the unbinding force comes from the interactions between PEI and DNA, we used a dsDNA-modified

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EXPERIMENTAL SECTION

Materials. PEI (branched, average Mw 25 kDa, Sigma-Aldrich), PBS tablet (Sigma-Aldrich, one tablet is dissolved in 200 mL of H2O to produce the 100% PBS buffer solution containing 137 mM of NaCl, 2.7 mM of KCl, 10 mM of Na2HPO4, 2 mM of KH2PO4, pH 7.4), primers (Sangon Biotech (Shanghai) Co., Ltd.), streptavidin (Promega), (3-aminopropyl)dimethylmethoxysilane (Fluorochem, UK), 21058


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Figure 4. Direct measurement of PEI/DNA interaction. (a) dsDNA-modified AFM tip was brought to interact with PEI-covered silicon surface. (b) Typical sawtooth curves. (c, d) Unbinding force distributions of sawtooth peaks obtained at pH 5.0 and pH 7.4, respectively.

Figure 5. Ionic strength dependence of unbinding force. The unbinding force measurement performed in (a, b) 10% PBS, I = 0.015; (c, d) 50% PBS, I = 0.075; (e, f) 100% PBS, I = 0.15, which produced the most probable forces of 24.9, 12.9, and 6.6 pN, respectively. 21059


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Figure 6. Unbinding force under different stretching speed. Unbinding force between dsDNA-modified AFM tip and PEI-functionalized silicon surface obtained at (a) 0.2, (b) 0.5, (c) 1, (d) 5, and (e) 10 μm/s. (f) The most probable unbinding forces show no obvious stretching speed dependence.

Table 1. Four Primers for the Preparation of Labeled dsDNA 2700 bp thiol, biotin labeled dsDNA 2700 bp amino labeled dsDNA

forward primer

reverse primer

5′-HS-(CH2)6-AGCGTGACACCACGATGC-3′ 5′-H2N-AGCGTGACACCACGATGC-3′

5′-biotin-CGCCACATAGCAGAACTT-3′ 5′-CGCCACATAGCAGAACTT-3′

DNA polymerase, 34.5 μL of ddH2O) in an Eppendorf thermal cycler (Applied Biosystems, EppendorfAG, Germany). Initially before 30 cycles, there was a hot start by heating the solution to 94 °C for 2 min. Each cycle comprised three steps: first, a denaturation step in which the solution was heated to 94 °C for 15 s; second, an annealing step for 30 s at 53 °C (a temperature of 10 °C below the average melting temperature of both primers); third, an extension phase that lasted 2 min 42 s at 68 °C. The product was checked by 0.7% agarose-TAE gel and purified with EasyPure PCR Purification Kit (TransGen Biotech, Beijing) and eluted with 10% PBS (pH 7.7). The DNA concentration was estimated by using a Helios UV−vis spectrophotometer (Thermo Electronics) at 260 nm. HPLC-pure primers used here were shown in Table 1.

N-hydroxysuccinimide (NHS)-biotin, 6-mercapto-1-hexanol (MCH), dimethyl sulfoxide (DMSO), and oxalic acid (Sigma-Aldrich) were used as received. Si3N4 tips (MSCT) were purchased from Bruker Nano, Santa Barbara, CA. Other chemicals, H2SO4 (AR), methanol (GR), alcohol (AR), and H2O2 (GR) were purchased from Beijing Chemical Works. Synthesis and Purification of dsDNA. Two kinds of 2700 bp double-stranded (ds) DNA (Table 1) were synthesized by polymerase chain reaction (PCR), using KOD kit purchased form Toyobo Co., Ltd. The reactions were carried out in 200 μL PCR tubes. Each tube contains 50 μL of reaction mixture as the final volume following the standard procedure of KOD Plus PCR Kit (5 μL of 10× reaction buffer, 5 μL of 2 mM dNTPs, 2 μL of 25 mM MgSO4, 1.5 μL of 10 μM primer mixture, 1 μL of 60 μg/mL pCERorid plasmid temple, 1 μL of 1U/μL KOD 21060


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ACS Applied Materials & Interfaces dsDNA-Functionalized Substrates. Au substrates were cut into 1.4 × 1.4 cm2 slices, after ultrasonic cleaning in piranha solution for 5 min, and washing by a large amount of water. Then, 40 μL of 6 μg/mL DNA (thiol and biotin labeled) solution (in 50 mM Tris pH 7.5) or dsDNA−PEI mixture in different N/P ratios (0, 0.75, 1.5, 3.0, 5.4) was added to the substrates and incubated overnight at 20 °C, and dsDNA could be linked to the substrate by S−Au bond. Before the SMFS experiment, the DNA sample was incubated with 1 mM 6-mercapto-1hexanol (MCH) PBS buffer to reduce the nonspecific adhesion between dsDNA and Au substrate. Biotin−Streptavidin-Functionalized Tip. The AFM tips were silanized according to the method established before.34,48 In brief, Si3N4 tips were cleaned with a mixture of 98% H2SO4 and 30% H2O2 (the volume ratio was 7:3, also known as piranha solution) for 2 min, and washed by distilled water. Then the tip was dried at 115 °C first, in an oven for 1.5 h, and put into a desiccator for cooling to room temperature. After that, 40 μL of (3-aminopropyl)dimethylmethoxysilane was added into the desiccator, and the tip was incubated at 25 °C for 1.5 h. Finally the AFM tip was washed in methanol, and dried at 115 °C for 10 min. Then the amino-silanized tip was incubated in 1 mM N-hydroxysuccinimide (NHS)-biotin in DMSO solution at 25 °C for 3 h. Before use, biotin-modified AFM tip was immersed in 1 mg/mL streptavidin in PBS buffer for 5 min, followed by rinsing with PBS buffer. dsDNA-Modified AFM Tip. Amino-silanized tips (prepared as before) were cultured with N-(3-dimethylaminopropyl)-N′-enthylcarbodiimide (EDC)/N-hydroxysucciinimide (NHS)/oxalic acid in PBS buffer (10 mg EDC, 10 mg NHS, 2.5 mg oxalic acid in every 100 μL PBS, pH 7.4) for 1 h at room temperature. Then, the AFM tip was incubated with 200 μg/mL of amino labeled dsDNA (in 10% PBS buffer pH 7.7) overnight. PEI-Functionalized Substrates. A silicon wafer was cut into 1 × 1 cm2 slices, and after ultrasonic cleaning in alcohol, the slices were then cleaned in boiling piranha solution for 20 min. After that, the silicon slices were washed, dried, and silanized as described above. The silanized slides were then cultured with EDC/NHS/oxalic acid in PBS buffer for 1 h at room temperature. Then, modified silicon slices were incubated in 5 ppm of PEI (in 10% PBS buffer pH 7.4) for 2 h and stored in 10% PBS solution (pH 5.0) before use. In order to check whether the substrates are covered by PEI molecules, the slides were imaged by AFM before and after culturing with 2 μg/mL dsDNA in TE (1 mM EDTA, 50 mM Tris, pH 7.5) buffer (Figure 7). As can be seen from Figure 7, DNA fragments appeared on the PEI-functionalized silicon surface (Figure 7b), which indicate that PEI has been attached to the silicon surface, since it is pretty difficult for

dsDNA to be immobilized on the pure silicon surface without the help of multivalent cations. AFM-Based Single Molecule Force Spectroscopy. In SMFS experiments a NanoWizardII BioAFM (JPK instrument AG, Berlin, Germany) was used in contact mode. The spring constants of AFM tips were calibrated by thermal noise method.49 The thiol-terminal of the dsDNA was immobilized on the Au substrate via thiol−gold chemistry leaving the biotin end available for the picking up by the streptavidinfunctionalized AFM tip. During the movement of the AFM tip away from the substrate, dsDNA−PEI complexes could be destroyed gradually by stretching, and the rupture events were recorded on the force− extension curve. In another case, the dsDNA-modified tip was brought into contact with the PEI-functionalized substrate; upon separation, the unbinding force between dsDNA and PEI could be recorded. Force− extension curves that show characteristic overstretching force plateaus at ∼65 pN (force fingerprint of a single dsDNA molecule, by which the single molecule signal can be identified) were analyzed. Unless stated otherwise, the single molecule force spectroscopy experiments were performed in 10% PBS. Unbinding forces were measured by using selfwritten macros embedded in Igor Pro, and histograms of unbinding forces were fitted by a Gaussian function to obtain the most probable unbinding forces.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by NNSFC (21525418, 21474041, 21221063, 20974039, 91127031), the National Basic Research Program (2013CB834503), and the Program for New Century Excellent Talents in University (NCET). We also thank the State Key Laboratory for funding.

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Figure 7. PEI-covered silicon wafer was imaged (a) before and (b) after incubation with 2 μg/mL of dsDNA by atomic force microscope in air. 21061


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Quantifying the Interactions between PEI and Double-Stranded DNA

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