Rationally Designed Sensing Selectivity and Sensitivity of an. Aerolysin Nanopore via Site-Directed Mutagenesis

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1 Rationally Designed Sensing Selectivity and Sensitivity of an Aerolysin Nanopore via Site-Directed Mutagenesis Ya-Qian Wang, Chan Cao, Yi-Lun Ying, Shuang Li, Ming-Bo Wang, Jin Huang, Yi-Tao Long*, Key Laboratory for Advanced Materials, School of Chemistry & Molecular Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai , P. R. China School of Pharmacy, East China University of Science and Technology, 130 Meilong Road, Shanghai , P. R. China * Table of Contents Experimental details Reagents and chemicals; proaerolysin production, formation of biological nanopores and data acquisition and analysis. Figure S1 Current-voltage curve and baseline at different voltages of WT aerolysin. Figure S2 Current-voltage curve and baseline at different voltages of R220E mutant aerolysin. Figure S3 Current-voltage curve and baseline at different voltages of K238E mutant aerolysin. Figure S4 Scatter plots of Poly(dA)4 by WT aerolysin at different voltages. Figure S5 Duration time histograms of Poly(dA)4 by WT aerolysin at different voltages. Figure S6 Scatter plots of Poly(dA)4 by R220E mutant aerolysin at different voltages. Figure S7 Duration time versus applied voltage of Poly(dA)4 by R220E mutant aerolysin. Figure S8 Scatter plots of Poly(dA)4 by K238E mutant aerolysin at different voltages. Figure S9

2 Duration time histograms of Poly(dA)4 by K238E mutant aerolysin at different voltages. Figure S10 Continuous time-current recording traces of Poly(dA)4 by K238E mutant aerolysin at voltage range from +80 mv to +160 mv. Figure S11 SDS-PAGE analysis of proaerolysin. Table 1 The percentage of current blockages in total events of WT and mutant aerolysin. Table 2 Duration time of WT and mutant aerolysin. Experimental details Reagents and chemicals Trypsin-EDTA, trypsin-agarose, decane (anhydrous, 99%), NaCl, Na3PO4, HCl and imidazole were purchased from Sigma-Aldrich Co., Ltd. (St. Louis, MO). 1, 2- Diphytanoyl-sn-glycero-3-phosphocholine (chloroform, 99%) was purchased from Avanti Polar Lipids, Inc. (Alabaster, Al, USA). All polynucleotides samples were synthesized and HPLC-purified by Sangon Biotech Co., Ltd. (Shanghai, China). All reagents and materials are of analytical grade. Yeast extract and peptone were purchased from OXOID Co., Ltd. (Basingstoke, UK). Glycerinum was purchased from Amresco, Inc. (Atlanta, GA, USA). IPTG were purchased from Inalco SpA, Inc. (Milano, Italy). BL21 [DE3] plyss E. coli was purchased from TIANGEN Co., Ltd. (Beijing, China). The pet22b-proaerolysin plasmid were synthesized and HPLC-purified by Genewiz, Inc. (Suzhou, China). All solutions were prepared using ultrapure water (18.2 MΩ cm at 25 C) from a Milli-Q system (Billerica, MA, USA). Proaerolysin production The gene of WT proaerolysin was retrieved from PDB (3G40) and synthesized by Genewiz, Inc. We used site-directed mutagenesis kit (TIANGEN Co., Ltd., Beijing, China) to introduce point mutations in plasmid DNA templates and then mutant Plasmid DNA was purified using a plasmid extraction kit (TIANGEN Co., Ltd., Beijing, China). Target gene was introduced to pet22b vector and BL21 [DE3] plyss E. coli were transformed with the pet22b-proaerolysin plasmid, which allows periplasmic expression of the toxin with a His6 tag on the C-terminus. BL21 [DE3] plyss E. coli harboring the WT aerolysin expression plasmid were grown at 37 C in 1 L cultures to an OD600 of 0.6. IPTG (0.25 mm) was added and the temperature was shifted to 16 C

3 for protein production. Cells were harvested after about 2 h and reaching an OD600 = 1.2. E. coil cells were collected from the LB medium by centrifugation (3000 rpm for 30 min at 4 C ). 50 ml buffer contained 0.5 M NaCl, 20 mm Na3PO4, ph 7.4 was added into the sediment and made it resuspended. After sonication on ice for 10 min, samples were centrifuged for 30 min at 4 C and rpm. Then the supernatant was load on to a Ni-NTA sorbent using gravity flow column. In order to remove the unspecifically bound proteins, the column was washed with buffer (20 mm Na3PO4, 0.5 M NaCl, ph 7.4) with a linear gradient of imidazole (0-0.5 M). The protein was further purified by size exclusion chromatography using a Superdex /600 PG column (GE Healthcare) equilibrated in 20 mm Tris-HCl, ph 7.4, and 500 mm NaCl. The purity of protein was confirmed by SDS-PAGE (Figure. S9). Equivoluminal 30% (v/v) glycerinum was added into the protein. At last, the proaerlysin was stored at -80 C. Formation of biological nanopores Monomeric aerolysin were acquired from proaerolysin by digesting with trypsin-edta for 1 h at room temperature. Lipid bilayers were formed from 1, 2-diphytanoyl-snglycero-3-phosphocholine (Avanti Polar Lipids) and spanning a 50 μm orifice in a Delrin bilayer cup (Warner Instruments, Hamden, CT). Both compartments of the recording chamber contained 1.0 ml of 1.0 M KCl, 10 mm Tris, ph 8.0, with 1.0 mm EDTA. The potential is applied using Ag/AgCl electrodes. About 1.0 µl monomeric aerolysin ( 1.5 µg/ml) was added to the cis chamber to form the pore. And polynucleotide was added to the cis compartment to a final concentration of 2.0 µm. All of the nanopore experiments were conducted at 24 ± 2 C. Data acquisition and analysis The current recordings were performed with a patch clamp amplifier (Axon 200B equipped with a Digidata 1440A A/D converter, Molecular Devices, USA) with the cis compartment connected to ground. The amplified signal (arising from the ionic current passing through the pore) was low-pass filtered at 5 khz and sampled at 100 khz by running the Clampex 10.4 software (Molecular Devices, USA). The data analysis was performed by using Mosaic software and Origin-Lab 8.0 (Origin-Lab Corporation, Northampton, MA)

4 Figure S1 Current-voltage curve (a) and baseline (b) at different voltages of WT aerolysin pore. An aerolysin pore inserted into a lipid membrane in the solution of 1.0 M KCl, 10 mm Tris, and 1.0 mm EDTA at ph 8.0. Figure S2 Current-voltage curve (a) and baseline (b) at different voltage of R220E mutant aerolysin pore. An R220E mutant aerolysin pore inserted into a lipid membrane in the solution of 1.0 M KCl, 10 mm Tris, and 1.0 mm EDTA at ph 8.0.

5 Figure S3 Current-voltage curve (a) and baseline (b) at different voltages of K238E mutant aerolysin pore. A K238E mutant aerolysin pore inserted into a lipid membrane in the solution of 1.0 M KCl, 10 mm Tris, and 1.0 mm EDTA at ph 8.0.

6 Figure S4 Scatter plots of Poly(dA)4 by WT aerolysin at potential of (a) 80 mv, (b) 100 mv, (c) 120 mv, (d) 140 mv and (e) 160 mv. The data were acquired in 1.0 M KCl, 10 mm Tris, 1.0 mm EDTA, ph 8.0 and in the presence of 2.0 µm Poly(dA)4.

7 Figure S5 Duration time histograms of Poly(dA)4 by WT aerolysin at potential of (a) 80 mv, (b) 100 mv, (c) 120 mv, (d) 140 mv and (e) 160 mv. All of the histograms were fitted to single Exponential function. The data were acquired in 1.0 M KCl, 10 mm Tris, 1.0 mm EDTA, ph 8.0 and in the presence of 2.0 µm Poly(dA)4.

8 Figure S6 Scatter plots of Poly(dA)4 by R220E mutant aerolysin at potential of (a) 80 mv, (b) 100 mv, (c) 120 mv, (d) 140 mv and (e) 160 mv. The data were acquired in 1.0 M KCl, 10 mm Tris, 1.0 mm EDTA, ph 8.0 and in the presence of 2.0 µm Poly(dA)4.

9 Figure S7 Duration time versus applied voltage of Poly(dA)4 by R220E mutant aerolysin. The data were acquired in 1.0 M KCl, 10 mm Tris, 1.0 mm EDTA, ph 8.0 and in the presence of 2.0 µm Poly(dA)4.

10 Figure S8 Scatter plots of Poly(dA)4 by K238E mutant aerolysin at potential of (a) 80 mv, (b) 100 mv, (c) 120 mv, (d) 140 mv and (e) 160 mv. The data were acquired in 1.0 M KCl, 10 mm Tris, 1.0 mm EDTA, ph 8.0 and in the presence of 2.0 µm Poly(dA)4.

11 Figure S9 Duration time histograms of Poly(dA)4 by K238E mutant aerolysin at potential of (a) 80 mv, (b) 100 mv, (c) 120 mv, (d) 140 mv and (e) 160 mv. All of the histograms were fitted to single Exponential function. The data were acquired in 1.0 M KCl, 10 mm Tris, 1.0 mm EDTA, ph 8.0 and in the presence of 2.0 µm Poly(dA)4.

12 Figure S10 Continuous time-current recording traces of Poly(dA)4 by K238E mutant aerolysin at potential of (a) 80 mv, (b) 100 mv, (c) 120 mv, (d) 140 mv and (e) 160 mv. The data were acquired in 1.0 M KCl, 10 mm Tris, 1.0 mm EDTA, ph 8.0 and in the presence of 2.0 µm Poly(dA)4.

13 Figure S11 SDS-PAGE analysis of WT and mutant proaerolysin. R220E mutant aerolysin (1),WT aerolysin (2), K238E mutant aerolysin (3). Table 1. The Percentage of Current Blockages in Total Events of WT and Mutant Aerolysin E1/Etotal (%)* +80 mv +100 mv +120 mv +140 mv +160 mv WT 47.1%±3.0% 59.5%±0.5% 71.3%±0.6% 78.4%±0.9% 81.8%±0.7% R220E 1.0%±0.7% 0.5%±0.4% 0.5%±0.2% 0.5%±0.2% 0.7%±0.1% K238E 39.6%±3.0% 56.0%±1.2% 72.2%±1.3% 82.2%±1.5% 89.0%±1.5% * E 1/E total: The percentage of current blockages (duration time longer than 0.13 ms, denoted as E 1) in total events (denoted as E total). Table 2. Duration time of WT and Mutant Aerolysin Duration time (ms) +80 mv +100 mv +120 mv +140 mv +160 mv WT 8.81± ± ± ± ±0.06 R220E* N. A. N. A. N. A. N. A. N. A. K238E 5.70± ± ± ± ±10.55 * There are barely no current blockages produced by R220E.