Letter pubs.acs.org/ac
Driven Translocation of Polynucleotides Through an Aerolysin Nanopore Chan Cao, Jie Yu, Ya-Qian Wang, Yi-Lun Ying, and Yi-Tao Long* Key Laboratory for Advanced Materials and Department of Chemistry, East China University of Science and Technology, Shanghai 200237, P. R. China S Supporting Information *
ABSTRACT: Aerolysin has been used as a biological nanopore for studying peptides, proteins, and oligosaccharides in the past two decades. Here, we report that wild-type aerolysin could be utilized for polynucleotide analysis. Driven a short polynucleotide of four nucleotides length through aerolysin occludes nearly 50% amplitude of the open pore current. Furthermore, the result of total internal reflection fluorescence measurement provides direct evidence for the driven translocation of single polynucleotide through aerolysin.
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with a constriction of 1.4 nm,17 aerolysin possesses the narrower bottlenecks of approximately 1 nm at its extracellular mouth and/or transmembrane β-barrel.18 This unique geometry may provide a high spatial resolution. Furthermore, aerolysin has seven unpaired positive charged residues in its lumen.18 These featured charge distributions can effectively enhance the interaction between negatively charged polynucleotide and aerolysin, resulting in a slow translocation speed for nucleic acid.19 In this paper, we demonstrate that the unlabeled random ssDNA could be detected by wild-type aerolysin with high current and temporal resolution (Figure 1a). Moreover, the total internal reflection fluorescence (TIRF) measurements provide a direct evidence to confirm the translocation of the random ssDNA through aerolysin.
anopore analysis is an emerging platform for single molecule analysis.1−4 The concept of nanopore sensing was first proposed in 1996 by using a biological nanopore, αhemolysin (α-HL). The basic principle of nanopore is based on monitoring the transient changes of ionic current produced by single polynucleotide through the pore.5 This technology provides a unique analytical capacity for identification and characterization of polynucleotides without sample amplification. The ultimate goal of most nanopore techniques is to achieve an inexpensive, high-throughput, and rapid singlemolecule DNA sequencing.6 Although α-HL has played a crucial role in the biological nanopore so far, other efficient biological nanopores are explored to improve the sensitivity and exploit the application of polynucleotide detection.7,8 For instance, Mycobacterium smegmatis porin A (MspA) owing to its short (∼0.5 nm long) and narrow (∼1 nm in diameter) constriction exhibits high sensitivity for discrimination of current levels caused by each base.9 Phi 29 motor protein10 is reported to expand the application of nanopore from single strand DNA (ssDNA) to double strand DNA. Therefore, it is very necessary and significant to explore the versatile poreforming materials for polynucleotide applications. Here, we introduce aerolysin as a prospective candidate for detection of nucleic acid at single-molecule level. Aerolysin is a heptameric pore-forming toxin from Aeromonas hydrophila. It allows spontaneous insertion into the lipid bilayer leading to a nanoscale pore which diameter ranges from 1.0 to 1.7 nm.11,12 In 2006, aerolysin was first used as a biological nanopore to study the α-helix peptides.13 Subsequently, it has been applied to study the dynamics of unfolded proteins14 and oligosaccharides15 and kinetics of enzymatic degradation.16 Compared to the wild-type α-HL © XXXX American Chemical Society
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EXPERIMENTAL SECTION Reagents and Chemicals. Trypsin-EDTA, trypsin-agarose, and decane (anhydrous, ≥99%) were purchased from SigmaAldrich Co., Ltd. (St. Louis, MO). Proaerolysin was purchased from Aerohead Scientific, Ltd. (Saskatoon, SK, Canada). 1,2Diphytanoyl-sn-glycero-3-phosphocholine (chloroform, ≥99%) was purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). All polynucleotides samples were synthesized and HPLCpurified by Sangon Biotech Co., Ltd. (Shanghai, China). All reagents and materials are of analytical grade. All solutions were prepared using ultrapure water (18.2 MΩ cm at 25 °C) from a Milli-Q system (Billerica, MA). Received: April 18, 2016 Accepted: April 27, 2016
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DOI: 10.1021/acs.analchem.6b01514 Anal. Chem. XXXX, XXX, XXX−XXX
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Analytical Chemistry
fluorescence emission was collected and imaged by using a NA = 1.40, 100× oil immersion objective.
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RESULTS AND DISCUSSION As shown in Figure 1a, an aerolysin protein is embedded in the lipid bilayer to form a nanopore from the cis side. According to 10 independent nanopore experiments, the current−voltage curves demonstrate that aerolysin has a good reproducibility (Figure S1). Besides, the structure of aerolysin is quite stable, which could keep a constant conductance even in the presence of 1 M denaturing agent of guanidium chloride. In our experiments, a short ssDNA (denoted as Oligo-1; sequencing, 5′-ACTG-3′) is designed to test the ability of aerolysin for polynucleotide detection. After addition of Oligo-1 in the cis chamber, large amounts of unambiguous current signals are observed and most of signals show a nearly 50% current blockage (Figure 1b and supplementary video). According to previous investigation in α-HL nanopore, the shortest length of the polynucleotide α-HL could be detected is a four base of polydeoxyadenylic acid.24 To compare the capacity of α-HL and aerolysin for detection of polynucleotide, we detected Oligo-1 by wild-type α-HL from both the cis and trans sides, no current signals were acquired (Figure S2). These results demonstrate that aerolysin has high current sensitivity and prolonged duration time for polynucleotides detection. The high sensitivity of aerolysin may owe to two reasons: (1) The narrower diameter of aerolysin provides a more confined space for the translocation of polynucleotide, producing obvious recognition of current signals compared with α-HL nanopore. (2) The charged amino acids in the lumen of aerolysin enhances the interaction between polynucleotide and aerolysin.18 Next, we statistically analyze the current signals produced by Oligo-1. The contour plots present that the current events originating from the interaction between Oligo-1 and aerolysin yield two populations (labeled as E1 and E2, Figure 2a−c). The events of E1 exhibit substantially longer duration time and more concentrated blockage current compared to that of E2. The histograms of Ires/I0 (Ires represents the residual pore current, while I0 stands for the open pore current) were fitted to the Gaussian distribution. The data shows that E1 is centered in Ires/I0 of 0.51, while E2 has a wide range of Ires/I0 from 0.6 to 0.9. The duration time of E1 displays a strong voltage dependence, whereas that of E2 remains consistent as the driven potential increasing from 80 to 140 mV (Figures S3− S5). Similar to previous studies of α-HL,24 we deduced that the E1 is produced by the translocation of Oligo-1 through aerolysin, whereas E2 caused by bumping events that a part of polynucleotides entering the aerolysin but eventually returned to the cis solution. Furthermore, the voltagedependent studies reveal that the duration time of E1 decreased exponentially with the applied voltage increasing from 80 to 140 mV (Figure 2d), which confirms the translocation of Oligo1 through the aerolysin. Notably, the percentage of E1 in total events decreased from 73% at 80 mV to 40% at 140 mV (Figure S6). These findings suggest that the increasing voltage accelerates the translocation of Oligo-1 through aerolysin but reduces the translocation probability. To obtain a direct evidence for the driven translocation of Oligo-1 through the aerolysin, we further design Oligo-2. A fluorescent moiety, Hexachloro-Fluorescein (HEX), is attached to the 5′ of Oligo-1. As illustrated in Figure 3a, Oligo-2 produces distinguishable current blockage signals by aerolysin
Figure 1. (a) Illustration of single Oligo-1 traversing through an aerolysin pore. The two compartments of the bilayer cell were termed cis and trans, and the Oligo-1 was presented in the cis solution. A positive potential was applied from the trans side by Ag/AgCl electrodes, with the cis solution grounded. Thus, the negatively charged polynucleotides translocate aerolysin from cis to trans side in the condition of 1.0 M KCl, 10 mM Tris, 1.0 mM EDTA, pH = 8.0. (b) Raw current recording traces for addition of Oligo-1 to the cis side of aerolysin at +100 mV. Compared to the absence of Oligo-1, large amounts of unambiguous current blockage signals are obtained with the aerolysin nanopore.
Formation of Biological Nanopores. Monomeric aerolysin were acquired from proaerolysin by digesting with trypsinEDTA for 1 h at room temperature. α-HL wild-type D8H6 was produced by expressing in BL21 (DE3) pLysS Escherichia coli cells and then purified by using a Ni column.20 The monomer and heptamer proteins were separated via 8% SDS-PAGE. The heptamer band was cut from the gel. The purified heptamer was conserved in 10 mM Tris-HCl and 1.0 mM EDTA at pH = 8.0 and stored in a −80 °C freezer. Nanopore Experiments and Data Analysis. The nanopore detection method was conducted according to our previous studies.21 The lipid bilayer membrane was formed spanning a 50 μm orifice in a Delrin bilayer cup (Warner Instruments, Hamden, CT). Unless otherwise stated, both compartments contain 1.0 mL of buffer (1.0 M KCl, 10 mM Tris, 1.0 mM EDTA, pH = 8.0). The aerolysin (1.0 μL, 1.5 μg mL−1) and the polynucleotides (2.0 μM in chamber) were added to the cis solution. All of the nanopore measurements were conducted at 24 ± 2 °C. The current traces were measured by a homemade amplifier (DFCA-001) equipped with a Digidata 1440A A/D converter (Molecular Devices, Forest City, CA). The signals were low-pass filtered at 5 kHz and acquired at the sampling rate of 100 kHz by running Clampex 10.4 software (Molecular Devices, Forest City, CA). The data analysis was performed by using homemade software22 and Mosaic software23 and OriginLab 8.0 (OriginLab Corporation, Northampton, MA). Total Internal Reflection Fluorescence Measurement. A Leica AM TIRF MC total internal reflection fluorescence microscope was used to observe the solution collected from the trans side of aerolysin before and after aerolysin nanopore experiments. Oligo-2 was excited with a 561 nm laser, and the B
DOI: 10.1021/acs.analchem.6b01514 Anal. Chem. XXXX, XXX, XXX−XXX
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Analytical Chemistry
solution in the absence of Oligo-2 (Figure 3c,d). Therefore, our results provide direct evidence that polynucleotides definitely translocate through the aerolysin nanopore which result in characteristic current signals.
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CONCLUSION In summary, we report the ability of aerolysin for detection of polynucleotide. Although aerolysin has been applied as a biological nanopore for two decades, the excellent capacity of aerolysin for polynucleotide detection has been overlooked. By attaching of fluorescent group HEX, we prove that single polynucleotide definitely passes through aerolysin. Compared with the commonly used biological nanopores, wild-type aerolysin exhibits instinct advantages for unmodified polynucleotide detection without any additional improvements. Our findings would open up a great application of aerolysin to nucleic acids analysis, such as DNA sequencing, DNA damage identifications, microRNA analysis, and other nanopore-based single molecule analyses. Furthermore, aerolysin holds great potential for direct analysis of native DNA.
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Figure 2. (a) Contour plots, (b) Ires/I0 histograms, and (c) duration histograms of Oligo-1 at 100 mV. (d) Duration time versus applied voltage for E1. A single-exponential function was used to fit the durations from 80 to 140 mV. The statistical analysis events were acquired from three independent nanopore experiments, and the number of current blockages in each experiment is 3 000 at least.
ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b01514. Current−voltage curves of aerolysin pore (Figure S1), detection of Oligo-1 with α-HL nanopore (Figure S2), contour plots of Oligo-1 at different voltage (Figure S3), E1 and E2 duration histograms of Oligo-1 at different voltages (Figures S4 and S5), and percentage of E1 in total events (NE1/NTotal%) at different voltage (Figure S6) (PDF) Video of the raw current recording traces of the addition of Oligo-1 from the cis side of aerolysin (AVI)
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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 supported by the National Basic Research Program of China (973 Program, Grant No. 2013CB733700) and the National Natural Science Foundation of China (Grant Nos. 21421004 and 21327807). Y.-T.L. is supported by the Chang Jiang Scholars Program.
Figure 3. (a) Raw current traces and (b) contour plots produced by Oligo-2 at 100 mV. The TIRF images of solution collected from trans side of aerolysin (c) in the absence and (d) presence of Oligo-2 after continuous single-channel electrical recording for 6 h. Only one aerolysin pore existed in the membrane during the entire recording process.
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DOI: 10.1021/acs.analchem.6b01514 Anal. Chem. XXXX, XXX, XXX−XXX
