Subscriber access provided by Kaohsiung Medical University
Letter
Identification of essential sensitive regions of the aerolysin nanopore for single oligonucleotide analysis Yaqian Wang, Mengyin Li, Hu Qiu, Chan Cao, Mingbo Wang, Xueyuan Wu, Jin Huang, Yi-Lun Ying, and Yi-Tao Long Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01473 • Publication Date (Web): 08 Jun 2018 Downloaded from http://pubs.acs.org on June 10, 2018
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry
Identification of essential sensitive regions of the aerolysin nanopore for single oligonucleotide analysis Ya-Qian Wanga, Meng-Yin Lia, Hu Qiub, Chan Caoa, Ming-Bo Wangc, Xue-Yuan Wua, Jin Huangc, YiLun Yinga*, Yi-Tao Longa* a. Key Laboratory for Advanced Materials & School of Chemistry and Molecular Engineering, East China University of Science and Technology, Shanghai, 200237, P. R. China. b. State Key Laboratory of Mechanics and Control of Mechanical Structures, Nanjing University of Aeronautics and Astronautics, Nanjing, 210016, P. R. China. c. School of Pharmacy, East China University of Science and Technology, Shanghai, 200237, P. R. China. †Correspondence to:
[email protected];
[email protected] Phone: +86-021-6425-2339 ABSTRACT: The aerolysin nanopore is an emerging platform for single molecule analysis which displays high spatial and temporal resolution for the discrimination of single nucleotides, identification of DNA base modification and analyzing the structural transition of DNAs. However, to overcome the challenge of achieving the ultimate goal of the widespread real analytical application, it is urgent to probe the sensing regions of the aerolysin to further improve the sensitivity. In this paper, we explore the sensing regions of the aerolysin nanopore by a series of well-designed mutant nanopore experiments combined with molecular dynamics simulations-based electrostatic analysis. The positively charged lumen-exposed Lys-238, identified as one of key sensing sites due to the presence of a deep valley in the electrostatic potentials, was replaced by different charged and sized amino acids. The results show that the translocation time of oligonucleotides through the nanopore can be readily modulated by the choice of the target amino acid at 238 site. In particular, a 7-fold slower translocation at a voltage bias of +120 mV is observed with respect to the wildtype aerolysin which provide a high resolution for methylated cytosine discrimination. We further determine that both the electrostatic properties and geometrical structure of the aerolysin nanopore are crucial to its sensing ability. These insights open ways for rationally designing the sensing mechanism of the aerolysin nanopore, thus providing a novel paradigm for nanopore sensing.
Nanopore shows great applications as a single molecule detection device and a biophysical model system because the ionic current recording traces it produces contain information about the identity, concentration, structure, and dynamics of target molecules.1-6 The principle of nanopore analysis method is based on monitoring the ionic current modulation as the target molecule occupies the nanometer-scale pore.1 Protein nanopores such as commonly used α-hemolysin (α-HL) 7-10 and MspA11, 12 have been under extensive researches for the single molecule analysis in the past decades due to their unique structure and high sensing capability. To further improve sensitivity of biological nanopores, a number of novel alternates, including ClyA,13 FhuA,14 CsgG,15 OmpG,16 NfpA and NfpB17 were proposed and tested. Meanwhile, engineered protein nanopores with various mutant were also designed for achieving specific sensing proposes. Recently, we found that wild-type (WT) aerolysin exhibited superior resolution for discrimination of oligonucleotides.18, 19 Moreover, the WT aerolysin also displayed exceptionally high detection sensitivity for other functional molecules, such as peptides,20, 21 proteins,22, 23 sugars24, 25 and PEG.26 Aerolysin is a heptameric β-pore-forming toxin which could insert into the cell membrane and potentially lead to cell death.27 Unlike the popular α-HL,28 the aerolysin heptamer generally shapes resembling more a rivet than a mushroom. It
lacks a vestibule,27 and has a longer effective β-barrel with a diameter as small as 1.0 to 1.7 nm.29 Another notable feature is that aerolysin has a high number of charged residues inside lumen.27 Previously, it was demonstrated that it was feasible to modulate the charge density or distribution of aerolysin pore to improve its biosensing performance.30, 31 Therefore, we reasonable suggest that the outstanding sensing capability of aerolysin is due to electrostatic properties and geometrical structure.18 To further improve the sensing ability of aerolysin, it’s imperative to carefully probe its key sensing regions, and in turn, to modulate its sensing ability by well-designed mutants. Herein, we identified the sensing regions of aerolysin by combining the mutant nanopore experiments and molecular dynamics (MD) simulations. As shown in Figure 1a, the electrostatic potential determined by MD simulation exhibited two regions where drastic potential changes occurred, indicating these two regions were essential for DNA translocation. Accordingly, we first replaced the positively charged Lysine-238 (K238) in one of the sensing region with neutral Phenylalanine (F), denoted as K238F. The DNA produced a notably shorter duration time in K238F than in WT (Figure 2b). It seemed that the lumen charge was the main factor dominating the confined aerolysin-DNA interactions. However, when we substituted K238 with neutral Glycine (G) containing no side chain (denoted as K238G), it is unexpected that DNA produced approx-
ACS Paragon Plus Environment
Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
imately seven times longer duration time than in WT (Figure 2b). Moreover, the K238G could further achieve discrimination of methylated cytosine in a heteronucleotide with such super temporal sensitivity which is impossible to achieve by WT aerolysin. In this paper, we determined two sensing regions of aerolysin, then further proved that K238 site greatly contributed to the temporal resolution for oligonucleotides detection.
Figure 1. Identification of sensitive regions of the aerolysin nanopore for oligonucleotides detection. (a) All-atom model of fulllength aerolysin nanopore system. The black line is electrostatic potential distribution along the WT aerolysin at +120 mV. The green and orange bands represent the sensing regions. The 238 sites are shown in purple. (b) Left: top views of WT (black), K238F (blue) and K238G mutant (red) aerolysin; Middle: structure of amino acids, Lys, Phe and Gly; Right: current traces of Poly(dA)4 detecting by WT (black), K238F (blue) and K238G mutant (red) aerolysin, respectively.
As illustrated in Figure 1a, we used the all-atom simulations to study the electrostatic potential in the aerolysin nanopore system. We constructed a nanopore containing a full-length WT aerolysin embedded into a lipid bilayer membrane and solvated in 1.0 M KCl solution. (Supporting Information) The electrostatic potential distribution exhibited two evident changes which were shown in orange and green. Since only two charged residues R220 and D222 locate in the orange band29, we consider that the electrostatic potential change attributes to these charged residues. In the green band, the positively charged K238, negatively charged E237 and E258 induced more dramatically electrostatic potential fluctuation. We speculate that the positively charged K238 is critical to the negatively charged oligonucleotides detection and explore its roles in this work. Further extensive studies are on the way to explore roles of other amino acids in these two regions such as R220 and D222 for DNA sensing.
To prove our idea, we designed and produced two mutated forms of aerolysin whose amino acids at the position 238 were substituted by phenylalanine (or glycine), named K238F (or K238G) (Supporting Information, Figures S1-S4). Since the pores are homoheptamers, the mutations appear in all 7 subunits. At our experimental condition of pH=8.0, the Lys is positively charged with a long side chain, the Gly is neutral without the side chain, and the Phe is also neutral with a benzene ring (Figure 1b). The raw current traces of Poly(dA)4 by three types of aerolysin were evidently different from each other (Figure 1b). The current-voltage curves and baselines by ten independent nanopore experiments demonstrated that only one conductance existed in reproducing the pore formation (Figure S5). However, whether the aerolysin could assemble into different conductance states like α-hemolysin need further study.32-34 The statistical analysis of Poly(dA)4 by three types of aerolysin at +120 mV are shown in Figure 2. The events were analyzed by the normalized blockade current (Ires/I0, Ires represents the residual current, while I0 is the open pore ionic current, so the Ires/I0 represents the current ratio of blockade) and duration time. Our previous study demonstrated that the bumping of oligonucleotides induced the independent duration time on the voltage.35 These bumping events originated from a part of oligonucleotides entering the aerolysin pore but eventually returned to the cis solution. Since the events with duration time shorter than 0.2 ms showed the constant value with the increasing voltage, we excluded these bumping events in our further analysis. The data of Poly(dA)4 by WT were reproduced according to our previous work.18 The raw current recording traces of Poly(dA)4 by WT and mutant aerolysin yielded distinguishable current blockade and duration (Figure 1b). The Ires/I0 value of Poly(dA)4 through K238F was about 0.49±0.09, less than that of WT about 0.51±0.01 (Figure 2a). The error represented standard deviation of three independent nanopore experiments. These results illustrated that the Poly(dA)4 has a greater degree of blockage current level in K238F mutant than in WT aerolysin. In the case of K238G mutant, the Ires/I0 value was about 0.54±0.01 (Figure 2a), greater than that by WT. The molar volume of residues order is Phe>Lys>Gly,36 therefore, the size probably plays an important role in blockage current level. The long duration time is beneficial for revealing the aerolysin-DNA interactions. The duration time of Poly(dA)4 by WT was about 5.4±1.4 ms, while by K238F was only 2.1±0.8 ms (Figure 2b). This result seemingly proved that the removal of positive charge in the lumen of areolysin could remarkably accelerate the speed of DNA translocation. However, the duration time by K238G was about 38.4±0.9 ms (Figure 2b), approximately seven times longer than that by WT. This fact suggests that the substitution of Lys with Gly could enhance the aerolysin-DNA interactions. Although Gly is a neutral amino acid with no side chain, there is a surprisingly longer duration time of DNA. Therefore, the electrostatic properties should not be the sole crucial factor for the high sensitivity, and the geometrical structure as well as the Van der Waals (vdW) force also matters. Further, voltage-dependent experiments were conducted to study the effects of voltages on duration time (Figures 2c, S6S9). The duration time by WT and K238F aerolysin in Figure 2c both decreased exponentially with increasing voltage. These findings demonstrated that increasing voltage accelerated the transport of DNA through the aerolysin nanopore.
ACS Paragon Plus Environment
Page 2 of 5
Page 3 of 5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry
Figure 2. Statistical analysis of Poly(dA)4 by WT and mutant aerolysin pores. (a) The Ires/I0 histograms of Poly(dA)4 at +120 mV by WT (black), K238F (blue) and K238G (red) aerolysin, respectively. The solid lines are Gaussian fits to the histograms. (b) The duration histograms of Poly(dA)4 at +120 mV by WT (black), K238F (blue) and K238G (red) aerolysin, respectively. The histograms of duration time are all fitted to exponential distributions. (c) Effects of the applied voltage on the duration time of Poly(dA)4 by WT and mutant aerolysin. The applied voltage ranging from +80 mV to +180 mV in 10-mV increments. The error-bars indicated standard deviation from data derived from three independent experiments. All the nanopore experimental data in this paper were acquired in 1.0 M KCl, 10 mM Tris, 1.0 mM EDTA at pH 8.0. The concentration of Poly(dA)4 is 2.0 µM. (d) Electrostatic potential along the aerolysin nanochannel, WT (black), K238F mutant (blue) and K238G mutant (red) aerolysin pore at +120 mV bias. The zero bias potential has only a qualitative meaning. Protein is overlaid geometrically faithfully.
Interestingly, the duration time curve for K238G was nonmonotonic, namely, divided into two regimes (Figure 2c). At low applied voltages (80
