Rationally Designed Sensing Selectivity and Sensitivity of an

Sensing Selectivity and Sensitivity of an Aerolysin Nanopore via Site-Directed Mutagenesis. Ya-Qian Wang† , Chan ... Publication Date (Web): Apr...
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Rationally Designed Sensing Selectivity and Sensitivity of an Aerolysin Nanopore via Site-Directed Mutagenesis Yaqian Wang, Chan Cao, Yi-Lun Ying, Shuang Li, Mingbo Wang, Jin Huang, and Yi-Tao Long ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b00021 • Publication Date (Web): 05 Apr 2018 Downloaded from http://pubs.acs.org on April 8, 2018

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Rationally Designed Sensing Selectivity and Sensitivity of an Aerolysin Nanopore via Site-Directed Mutagenesis Ya-Qian Wang1‡, Chan Cao1‡, Yi-Lun Ying1, Shuang Li1, Ming-Bo Wang2, Jin Huang2, Yi-Tao Long1*

1

Key Laboratory for Advanced Materials & School of Chemistry and Molecular Engineering, East China University of Science and Technology, Shanghai, 200237, P. R. China. 2 School of Pharmacy, East China University of Science and Technology, Shanghai 200237, P. R. China. KEYWORDS: selectivity, sensitivity, single molecule, aerolysin, nanosensor ABSTRACT: Selectivity and sensitivity are two key parameters utilized to describe the performance of a sensor. In order to investigate selectivity and sensitivity of the aerolysin nanosensor, we manipulated its surface charge at different locations via single sitedirected mutagenesis. To study the selectivity, we replaced the positively charged R220 at the entrance of the pore with negatively charged Glutamic acid, resulting in barely no current blockages for sensing negatively charged oligonucleotides. For the sensitivity, we substituted the positively charged lumen-exposed amino acid K238 located at trans-ward third of the β-barrel stem with Glutamic acid. This leads to a surprisingly longer duration time at + 140 mV, which is about 20 times slower in translocation speed for Poly(dA)4 compared to that of wild-type aerolysin, indicating the stronger pore-analyte interactions and enhanced sensitivity. Therefore, it’s both feasible and understandable to rationally design specific biological nanosensors for single molecule detection with high selectivity and sensitivity.

To achieve the requirement of sensing, an excellent sensor must possess high selectivity and sensitivity simultaneously. Biological nanopore as a remarkable nanosensor, displays superb selectivity and sensitivity. Therefore, biological nanopore is used to identify and analyze single biomolecule by recording the changes of the ion current blocked by target analytes.1 In addition, its inherent merits such as simplicity, label-free detection, high resolution, and short detection time make biological nanopore sensor a promising technique for single-molecule analysis.2-4 However, to meet the increasing demand of high resolution, the sensing selectivity and sensitivity of wild-type biological nanopores need to be dramatically improved. Fortunately, the site-directed mutagenesis permits precise modification of protein nanopores so as to modulate the selectivity and sensitivity. For example, previous results reported that the manipulation of charge on the internal surface of α-hemolysin (α-HL) nanopore could improve the capture rate of DNA.5 And it was also reported that the exchange of negatively charged residues for positively charged residues in the vestibule region of MspA nanopore exhibited extraordinarily high pore-DNA interaction rates.6 However, in comparison with the widely used transmembrane proteins α-HL7, 8 and MspA,9, 10 the other pores such as aerolysin has narrower diameter, longer effective β-barrel, larger cap domain and lacks the vestibule.11-14 Besides, another excellent feature is 7 unpaired positively charged amino acids locating in its lumen.15 These compelling characteristics indeed provide a high resolution for the single molecule detection, such as peptides,16, 17 proteins,18-20 chimaera molecules,21 oligosaccharides,22

poly(ethylene glycol) (PEG) oligomers,23 and nucleic acids.2428 In addition, aerolysin is ideal for engineering via sitedirected mutagenesis, and it retains high levels of expression and channel-forming ability for a wide variety of amino acid replacements which provide the possibility to realize the single-site modification on its internal surface.29 Although the crystallization of aerolysin is still a challenge, the cryoelectron microscopy (cryo-EM) results showed that aerolysin was ultrastable.14 By combining our previous works, we found that there were two important positively charged amino acids R220 and K238 located at the entrance of the pore and the bottom of the lumen which produced high electrostatic interactions.27 We speculated that these two sites influenced the selectivity and sensitivity, respectively. To prove our idea, in this paper, we replaced the positively charged amino acids R220 and K238 with negatively charged Glutamic acid, denoted as R220E and K238E, respectively. As a result, negatively charged oligonucleotides produced barely no current blockages in R220E mutant aerolysin, indicating a high entry barrier. Moreover, the surprising long duration time of the oligonucleotide by the K238E mutant revealed a strong poreanalyte interaction, indicating that K238E had a higher sensitivity than wild-type (WT) aerolysin. These findings demonstrated that the R220 was related to the selectivity, while the K238 was responsible for the sensitivity. Further, we could rationally design highly selective and sensitive aerolysin nanosensors to achieve single molecule reactions and even simultaneous detection of multiple analytes.

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Figure 1. Illustration of Poly(dA)4 traversing through wild-type and mutant aerolysin pores. (a) Monomeric aerolysin self-assembled and then inserted into the membrane, the R220 were shown in red and K238 were shown in blue. (b) Examples of analyzing Poly(dA)4 through a single aerolysin nanopore. The WT aerolysin with positively charged amino acids Arg and Lys produced distinguishable current blockages (top). R220E mutant aerolysin with negatively charged Glu locating at the entrance of the pore, which barely produced the current blockages with the continuous time-current recording (middle). K238E mutant aerolysin with negatively charged Glu at the trans-ward third of the β-barrel stem lead to a prolonged duration. The data were acquired in 1.0 M KCl, 10 mM Tris and 1.0 mM EDTA at pH 8.0 and + 100 mV in the presence of 2.0 µM Poly(dA)4.

As shown in Figure 1, an aerolysin heptamer inserts into the lipid bilayer to form a nanopore from the cis side, and the potential is applied using Ag/AgCl electrodes. According to five independent nanopore experiments, the current-voltage curves indicated that aerolysin owned a good reproducibility (Figure S1-S3). The continuous time-current recording traces of baseline at different voltages further demonstrated that the structure of aerolysin was quite stable and could keep a constant conductance in our experimental condition (Figure S1-S3). The negatively charged oligonucleotides could be captured by the positively charged amino acids at the top of the aerolysin at first, then they interact with the amino acids in the lumen, especially with positively charged amino acids. Therefore, we consider that the cap domain of an aerolysin is responsible for capturing of the single oligonucleotide, while the β-barrel region governs the translocation process. On the basis of our previous research,27 we speculated the positively charged amino acids R220 and K238 which were located at the entrance and the lumen bottom of the aerolysin were of great importance. To study selectivity and sensitivity of aerolysin, we replaced the positively charged R220 and K238 with negatively charged Glutamic acid, respectively. The pores are homoheptamers, so the mutations appear in all 7 subunits. Then examinations of oligonucleotide (sequencing, 5’-AAAA3’) by WT and mutant aerolysin nanopores were conducted. We statistically analyzed the current signals produced by WT and mutant aerolysin at + 100 mV. The contour plots illustrate that the current events originate from the interaction between Poly(dA)4 and aerolysin. 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 duration time histograms were fitted to exponential distribution (WT aerolysin and K238E mutant) and single Gaussian distribution (R220E mutant). As shown in Figure 2a, the cur-

rent events by WT aerolysin exhibited concentrated Ires/I0 value and substantially long duration time which reflected both high spatial and temporal resolution. The fitted data showed that the peak was centered in Ires/I0 of 0.50 ± 0.02, and the duration time was about 6.68 ± 0.10 ms. However, the continuous time-current recording of Poly(dA)4 by R220E displayed barely no current blockages, and the data showed that Ires/I0 value had a wide range from 0.4 to 0.8 at + 100 mV with an extremely short duration time 0.08 ± 0.01 ms (Figure 2b). The experimental results from + 80 mV to + 160 mV showed that the duration time of Poly(dA)4 by R220E were shorter than 0.13 ms (which is the rise time for the amplifier at 5 KHz filter) (Figure S7). These findings demonstrated that the events we observed were bumping signals caused by a part of oligonucleotides entering the aerolysin but eventually returning to the cis solution, indicating a high entry energy barrier for sensing oligonucleotide molecules. This phenomenon is probably resulted from the repulsion between negatively charged amino acid Glu and the negatively charged oligonucleotide. The Figure 2c showed that Ires/I0 value of Poly(dA)4 by K238E was about 0.52 ± 0.01 which was comparable to that of WT, indicating that there might have little structural difference between WT and K238E. The duration time was 18.14 ± 0.50 ms, approximately three times longer than that of WT, revealing a stronger pore-analyte interaction. The signals exhibited substantial deep blockages with spike-like shape. The statistically analysis of second level signals by K238E exhibited two distributions of the blockage current level which may be related to the interaction between the pore and the oligonucleotide with dynamic conformation in the super-confined aerolysin nanopore (Figure 2d). Besides, with the applied voltage increasing, the frequency of second level signals increased (Figure S10). This phenomenon maybe resulted from the back

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Figure 2. 2D contour plots, related Ires/I0 and duration histograms of Poly(dA)4 by WT areolysin (a), R220E mutant (b) , K238E mutant (c) and second level signals by K238E (d). The 2D contour plots present the both current and duration information, also display the events density distribution. The histograms provide the statistical values of Ires/I0 and duration time. The populations of events in the 2D contour plots calculated were shown in the light blue rectangle. Ires/I0 histograms of Poly(dA)4 by WT aerolysin, R220E and K238E mutant were fitted to single Gaussian peak. Duration time histograms by WT aerolysin and K238E mutant were fitted to exponential distribution. Duration time histograms by R220E mutant were fitted to single Gaussian peak. The second level signals by K238E were shown in Figure 2d. The data were acquired in 1.0 M KCl, 10 mM Tris, 1.0 mM EDTA, pH 8.0 and at + 100 mV in the presence of 2.0 µM Poly(dA)4.

and forth motion of Poly(dA)4 caused by the interaction with negatively charged Glutamic acid. The findings mentioned above demonstrate R220E has high selectivity, K238E is ultrasensitive (the detailed semiquantitative values are shown in Supporting Information, Table 1 and Table 2). To further understand the results, the voltage dependent experiments were conducted (detailed experimental data could be found in Supporting Information). The results of Poly(dA)4 by K238E mutant aerolysin exhibited notably longer duration time (Figure 3a, S9), which increased with increasing voltage. This phenomenon is the result of the electric driving force we

applied is not able to overcome the energy barrier caused by the interaction between the analyte and pore at this position. Accordingly, we achieved high sensitivity for oligonucleotides detection by K238E mutant aerolysin. The effects of the applied voltage on the frequency displayed that there were no obvious difference between WT aerolysin and K238E mutant (Figure 3b). Combining all the results, we indeed demonstrated that the cap domain influenced the capture ability of the single oligonucleotide, while the β-barrel region dominated the translocation speed.

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cept will guide us to achieve the goal of advancing sensing selectivity and sensitivity.

ASSOCIATED CONTENT Supporting Information Experimental details: reagents and chemicals, proaerolysin production, formation of biological nanopores and data acquisition and analysis. Current-voltage curve and baseline at different voltages of WT aerolysin (Figure S1), current-voltage curve and baseline at different voltages of R220E mutant aerolysin (Figure S2), current-voltage curve and baseline at different voltages of K238E mutant aerolysin (Figure S3), scatter plots of Poly(dA)4 by WT aerolysin at different voltages (Figure S4), duration time histograms of Poly(dA)4 by WT aerolysin at different voltages (Figure S5), scatter plots of Poly(dA)4 by R220E mutant aerolysin at different voltages (Figure S6), duration time versus applied voltage of Poly(dA)4 by R220E mutant aerolysin (Figure S7), scatter plots of Poly(dA)4 by K238E mutant aerolysin at different voltages (Figure S8), duration time histograms of Poly(dA)4 by K238E mutant aerolysin at different voltages (Figure S9), continuous time-current recording traces of Poly(dA)4 by K238E mutant aerolysin at voltage range from + 80 mV to + 160 mV (Figure S10), SDS-PAGE analysis of proaerolysin (Figure S11), the percentage of current blockages in total events of WT and mutant aerolysin (Table 1), duration time of WT and mutant aerolysin (Table 2). The Supporting Information is available free of charge on the ACS Publications website. Figure 3. Effects of the applied voltage on the duration time (a) and frequency (b) of Poly(dA)4 by WT areolysin and K238E mutant in the condition of 1.0 M KCl, 10 mM Tris, 1.0 mM EDTA, pH = 8.0, the concentration of Poly(dA)4 is 2.0 µM in the cis chamber.

In summary, the positively charged R220 amino acid at the entrance of aerolysin nanosensor is related to selectivity, which controls whether the oligonucleotide could enter the pore. The positively charged K238 situated at the trans-ward third of the lumen is responsible for the sensitivity, which could govern the translocation speed. Our results also reflected that the DNA-pore interactions and charge decoration inside the pore were important to dominate the DNA translocation kinetics which had been reported by Payet et al..30 Therefore, the precise manipulation of the properties of biological nanopore by single-site modification makes it possible to meet the increasing sensing requirement. Previously, Xi et al. reported a powerful approach to achieve ultrasensitive detection of DNA in cancer cells by adding a salt gradient in the aerolysin nanopore system.31 Additionally, the characteristics of the analyte, including charge, structure, flexibility, size, as well as electrolyte nature and the applied voltage polarity and magnitude, all contribute to the electroosmosis and electrophoresis in the dynamics of biomolecules through biological nanopores.32 In the future, we could design specific biological nanosensor with high resolution and optimize the experimental conditions to directly detect single-nucleotide variations, epigenetic modifications, DNA damage, examine the activity of nucleases, and even make it feasible to study single molecule reaction kinetics. Further, as a reasonable model, the channel surface of a single membrane protein could be regarded as a single molecule interface, the design of single sensing zone at single molecule interface could ensure high spatial and temporal resolution towards single molecule analysis. This con-

AUTHOR INFORMATION Corresponding Author * Correspondence to: [email protected] Notes The authors declare no competing financial interest.

Author Contributions Y.-T.L., Y.Q.W., C.C., Y.-L.Y conceived the idea; Y.Q.W., C.C. designed the experiments; Y.Q.W. performed the nanopore experiments and data analysis; Y.Q.W., C.C., J.H. designed the mutant aerolysin; Y.Q.W., S.L., M.B.W. purified the WT and mutant aerolysin; Y.-T.L., Y.-L.Y., Y.Q.W, interpreted the data; Y.Q.W., Y.-L.Y., Y.-T.L. cowrote the paper; Y.-T.L., Y.-L.Y., C.C. supervised the project. / All authors have given approval to the final version of the manuscript. / ‡ These authors contributed equally.

ACKNOWLEDGMENT This research was supported by the National Key R&D Program of China (2017YFC0906500), National Natural Science Foundation of China (21421004 and 21327807), the “Chen Guang” project from Shanghai Municipal Education Commission and Shanghai Education Development Foundation, and the Fundamental Research Funds for the Central Universities (222201718001, 222201717003, 222201714012).

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