Recognizing the Translocation Signals of Individual. Peptide-Oligonucleotide Conjugates Using an α-hemolysin Nanopore - PDF

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Recognizing the Translocation Signals of Individual Peptide-Oligonucleotide Conjugates Using an α-hemolysin Nanopore Yi-Lun Ying a, Da-Wei Li a, Yu Lui a, Subrata K. Dey b, Heinz-Bernhard Kraatz c, Yi-Tao

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Recognizing the Translocation Signals of Individual Peptide-Oligonucleotide Conjugates Using an α-hemolysin Nanopore Yi-Lun Ying a, Da-Wei Li a, Yu Lui a, Subrata K. Dey b, Heinz-Bernhard Kraatz c, Yi-Tao Long a * a State Key Laboratory of Bioreactor Engineering & Department of Chemistry, East China University of Science and Technology, 130 Meilong Road, Shanghai, , P.R. China. b Department of Chemistry, Bankura Sammilani College, Kenduadihi, Bankura , WB, India. c Department of Physical and Environmental Sciences, University of Toronto Scarborough, 1265 Military Trail, Toronto, M1C 1A4, Canada. These authors contributed equally to this work Supplementary Information 1. Single-channel recording α-hl was purchased from Sigma-Aldrich (St. Louis, MO, USA) and used without purification. Diphytanoyl-phosphatidyl-choline was purchased from Avanti Polar Lipids Inc. (Alabaster, AL, USA). The final concentration of the analyte in 1 ml cis chamber is 0.1 μm. Unless otherwise noted, all other chemicals were of analytical grade. The lipid bilayers were created by applying diphytanoyl-phosphatidyl-choline (30 mg/ml) in decane (99%, Sigma-Aldrich St. Louis, MO. USA) to a 150 μm orifice in a 1 ml bilayer cup integrated into a lipid bilayer chamber (Warner Instruments, Hamden, CT, USA) filled with KCl (1.0 M) and Tris-HCl (10 mm, ph = 7.80). A bilayer was deemed stable by monitoring its resistance and capacitance. The two compartments of the bilayer cell are termed cis and trans, shown in Fig.1a, where the trans compartment is defined as virtual ground. The potential was applied from the cis side by an Ag/AgCl electrode. The experiments were run under voltage-clamp condition using a ChemClamp (Dagan Corporation, Minneapolis, MN, USA) instrument. Currents were filtered at 10 khz by DigiData 1440A (Axon Instruments, Forest City, CA, USA) hardware and recorded by a PC running PClamp 10.2 (Axon Instruments, Forest City, CA, USA). 1 The α-hl was injected adjacent to the aperture in the cis chamber, and pore insertion was determined by a well-defined jump in current value. Once a stable single-pore insertion was detected, analyte solution was added to the cis chamber, proximal to the aperture. Data was collected using a threshold level that was 20 pa from the baseline open channel current. Analysis of all data was performed by ClampFit 10.2 (Axon Instruments, Forest City, CA, USA) and OriginLab 8.0 (OriginLab Corporation, Northampton, MA, USA). The raw current data events were analyzed by measuring both the magnitude of the ionic current blockage and the duration of the current blockage on a home designed software. Thus, each event consists of two data points described as blockage current magnitude and duration. Based on previous literature, [1] the blockage of ionic flow under applied potentials is the result of biopolymers transiting the pore, resulting in a restriction in ionic flow. Reported standard deviations are based on the three separate experiments. 2. Analyzing of compound 1 and P7 using an α-hl pore Compound 1 displays a typical spectrum of collagen with a maximum at 226 nm, a minimum at 206 nm, and a crossover at 219 nm. The peptide (P7) used in this study exhibited a high propensity for triple-helix formation. [2] The circular dichroism (CD) spectral data of compound 1 is comparable to the control compound (Gly-Pro-Hyp) 7 -Cys (P7), as shown in Fig.S1. To confirm the triplex formation of collagen in compound 1, we used the ratio of the positive peak intensity over negative peak intensity, R pn. For compound 1 and P7, the R pn values are 0.22 and 0.17, respectively. These results indicate the presence of triple-helical conformations is established for compound 1 and control compound P7 at 20 C and ph = As the solution temperature gradually increased from 15 C to 90 C, the CD spectrum for compound 1 changed gradually, shows a pattern featuring the triple helix polypeptide in compound 1 with slight damage to the triple helix at higher temperature. The resulting T m value is 56 ± 2 C providing an indication of the conformational stability of triple helix for compound 1(Fig. S1c). We investigated the translocation of the P7 peptide through an α-hl nanopore and were able to discriminate the different degrees of association of P7 in real time based on the characteristic current signatures of individual translocation events. A P7 molecule was driven into α-hl from the cis side to the trans side of the pore at mv, resulting in a pulse-like signal (Fig.S2a). The current blockages associated with the translocation events fall into two populations positioned at 0.76 ± pa (PI), with half-peak width of 0.12 ± 0.07 pa, and 0.55 ± 0.02 pa (PII), with half-peak width of 0.09 ± 0.03 pa, as shown in Fig.S2b,c. These peaks are related to the degree of peptide association. The assignments of the current blockage peaks of P7 are similar to those that have been reported previously. [3] Thus, the translocation events of PI are assigned to P7 passing through the pore as a triple helix, whereas the events of PII are assigned to the collisions or the translocations of the dimer/monomer of P7. Most of the events fall into PI, with a lifetime of 0.44 ± 0.07 ms (Fig.S2d), indicating that P7 is prone to adopt a triple helical structure. The events in PII have a lifetime of 0.15 ± 0.03 ms, as illustrated in Fig.S2e. Fig.S1 (a) The CD spectrum of 0.05 mm compound 1 in an aqueous solution at 20 C and ph = (b) The CD spectrum of 1.20 mm P7 in an aqueous solution at 20 C and ph = The ratio of the positive peak intensity over the negative peak intensity (R pn ) could be used to establish the triplex formation of the analyte. For compound 1 and P7, the values of R pn are 0.22 and 0.13, respectively. These R pn values indicate that compound 1 adopts a collagen-like helix conformation. (c) Thermal denaturation of 0.05 mm compound 1 at 226 nm. Ellipticities at 226 nm were monitored by CD spectroscopy as the temperature was increased by increment of 5 C with 2 min equilibrium. 3 Fig.S2 Detection of P7 through an -HL pore in the presence of Tris-HCl ph = (a) Current traces of P7 events. Each signal has one current level. (b) Counter plot of the current transients of P7. (c) The histogram of the blockage current fits to two Gaussian peaks, labeled PI and PII. (d) A single exponential function was fitted to the histogram of the duration of the events in PI. The values of i/i 0 for the events in the histogram range from 0.64 to (e) Histogram of the duration for the events in PII fits by a Gaussian function. The values of i/i 0 for the events in the histogram range from 0.46 to Analyzing poly(da) 20 using an α-hl pore An individual poly(da) 20 molecule driven into an α-hl at mv produced a current trace with a single current level, as illustrated in Fig.S3a. The scatter plots of poly(da) 20 (Fig.S3b) suggest that the population of the short events at low blockage currents might be assigned to bumping events, and the long events at the high blockage currents might be related to translocations. The duration of the events was fitted to a Gaussian function and exhibited a peak value of 0.36 ± 0.03 ms. 4 Fig.S3 Monitoring the translocation of poly(da) 20 through an α-hl nanopore in a solution of ph=7.8 Tris-HCl at mv. (a) Current traces of poly(da) 20 events. Each poly(da) 20 molecule enters the α-hl pore, which results in a pulse-like signal. (b) Scatter plot of the blockage duration. (c) Gaussian function fit to the duration histogram of poly(da) 20. The fitted value of duration is 0.36 ± 0.03 ms. 4. Analyzing compound 2 using an α-hl pore These nanopore results confirm that a conjugate traversed through α-hl after being captured by the constricted section of the pore. The duration (t D ) for the entire translocation process was fitted to a Gaussian function (Fig.S4c), which suggested that the events in the typical three-segment shape correspond to the deterministic translocation of compound 2. The values of t I, t III and t D are 0.50 ± 0.04 ms, 0.61 ± 0.06 ms and 1.31 ± 0.05, respectively. After one oligonucleotide component entered into the vestibule, the other two poly(da) 60 would induce the large steric hindrance at the entrance of α-hl nanopore comparing to B20 in compound 1. Therefore, the translocation process of P7 encounter hindrance resulting in the value of t III for compound 2. 5 Fig.S4 Detection of compound 2 in a collagen-like helical conformation transiting through an α-hl pore in ph=7.80 Tris-HCl. The duration histograms of segment I (a), segment III (b) and the entire duration for three-segment events of compound 1 (c) at mv. An exponential function was fitted to the histogram of t I. The shape of the t III histogram is divided into two parts: for times less than the time associated with the peak value, the curve follows a relatively steep rise, but for times greater than the time associated with the peak value, the distribution is well approximated by an exponential decay, and the fitted value is 0.59 ± 0.06 ms. The histograms for t D were fitted to Gaussian functions. Each segment of the blockage current was fitted to a Gaussian function in (a) i I, (b) i II and (c) i III, respectively. 6 5. Analyzing poly(da) 60 using an α-hl pore Fig.S5 Detection of poly(da) 60 by the α-hl nanopore in a solution of Tris-HCl ph=7.80 at mv. (a) Scatter plot of current transients of poly(da) 60. (b) Histogram of the blockage current. A Gaussian function fitted to the duration histogram, and the fitted value of duration is 0.45 ± 0.05 ms. 6. Translocation of compound 3 through an α-hl pore Compound 3 were synthesized by conjugating B20 to another B20 instead of colleagen-like p7 (Fig.S6). At the applied potential of mv, a minority of events induced by driven compound 3 through α-hl fall into population III (PIII), as illustrated in Fig.S7a. The distribution of the blockage events reveals two distinct populations, PIV at 0.60 ± 0.03 and PV at 0.30 ± 0.03 (Fig.S7b). The number of events located in PV decreased with the potential increasing from mv to mv. These events correspond to the bumping and collisions of compound 3 (Fig.S7a-f). The number of events in PIII increased when the applied potential was increased. The hydrogen bonds between base pairs might exist between two compound 3 molecules because of the flexible poly(ethylene glycol) 3 linkage. Thus, compound 3 undergoes an unzipping process before translocation, which contributes to the blockages corresponding to PIV and PIII. Interestingly, the percentage of events with the three-segment signature among all the events increased with the positive potential. These percentages are ~ 2% at mv, ~ 5% at mv and ~ 20% at mv. At a potential of mv, the durations of S I were fitted to a two-exponential function, giving the values of 0.52 ± 0.05 ms and 5.18 ± 0.26 ms (Fig.S8a). Although the high potential will limit the hydrogen bonds between B20, it is impossible that each compound 3 adopts the linear form in the bulk solution. The fitting of t I suggests the linear and folded form of compound 3 co-exist in the solution. The small duration of S I attribute to the translocation of linear form while the large duration is appointed to the unfolding process of compound 3 before the translocation. Gaussian fitting of the 7 experimental results gave the value of t III = 0.45 ± 0.05 ms (Fig.S8b), which suggests a deterministic translocation. It is important to note that both PIV and PIII have three-segment signatures. The values of the blockage current for the three-segment events at mv were 0.69 ± 0.03, 0.50± 0.02 and 0.70 ± 0.03, respectively (Fig.8f-g). Therefore, the signals with flat bottoms in PIII and PIV might be attributable to the collisions of compound 3 with the α-hl. Previous studies indicated that oligonucleotides could traverse through α-hl from the cis side to the trans side. 4 Besides, the poly(ethyleneglycol) polymer was driven into α-hl and proved to decrease the single-channel conductance Thus, The typical three-segment events confirming the reproducibility of the three-segment signals which strongly indicates the translocation of compound 1 and 2. Fig.S6 The structure of compound 3. Two 20-mer oligonucleotides were conjugated using poly(ethylene glycol) 3 and this compound is denoted compound 3. 8 Fig.S7 Detection of the compound 3 through an α-hl pore in ph=7.80 Tris-HCl. (a) Scatter plot of compound 3 at mv. Insert: the three-segment signal observed at mv. (b) Histogram of the blockage currents of compound 3 at mv. The fitted values for the blockage current were i/i 0 = 0.60 ± 0.03 for PIV and i/i 0 = 0.30 ± 0.03 for PV. (c) Scatter plot of compound 3 at mv. Insert: the representative three-segment event. (d) The blockage current distribution of compound 3 at mv. The fitted values for the two Gaussian peaks were i/i 0 = 0.84 ± 0.02 for PIII and i/i 0 = 0.65 ± 0.04 for PIV. (e) Scatter plot of compound 3 at mv. Insert: typical three-segment signal obtained at mv. (f) Histogram for the blockage currents of compound 3 at mv. The fitted values for PIII and PIV were i/i 0 = 0.68 ± 0.04 and i/i 0 = 0.80 ± 0.05, respectively. The regions of PIII, PIV and PV in the scatter plots are framed in the blue box, red box and green box, respectively. 9 Fig.S8 The duration histograms of typical three-segment events for compound 3 at mv. (a) t I, (b) t II, and (c) t D. A two-exponential function was used to fit the histogram of t I. The histograms for t III were fitted into Gaussian functions. The distributions for t D are divided into two parts: for times less than the time corresponding to the peak value, the curve follows a relatively steep rise, and for times greater than the time corresponding to the peak value, the curve is well approximated by an exponential decay, and the fitted value is 1.46 ± 0.12 ms. Each segment of the blockage current was fitted to a Gaussian function in (d) i I, (e) i II and (f) i III, respectively. 7. Analyzing compound 4 using an α-hl pore Compound 4 which comprises two P7 segments as illustrated in Fig.S9, was synthesized and driven into the α-hl. The addition of compound 4 into the cis side of the nanopore produced short-lived current blockages at mv (Fig.S10a). The majority of events fall into one population at i/i 0 = 0.56 ± 0.04, with a peak duration of 0.45± 0.03 ms, as shown in Fig.S10b-d. When the potentials were increased from mv to mv, no typical three-segment signal appeared, and only a pulse-like signal was observed (Fig.S10a,e). A new population was found with the peak value of 0.25 ± 0.04 at mv (Fig.S10g), which indicates that a compound 4 molecule is prone to bump at the vestibule of the α-hl pore. The duration time of the events remains approximately constant with increasing potential from mv to mv (Fig.S10d,h). These results suggest that once the positively charged compound 4 enters the vestibule of the α-hl, it 10 frequently exits the nanopore from the cis side rather than traverses the nanopore. Thus, in comparison with compound 1 and compound 4, the B20 component is prior to enter the cavity of α-hl to ensure the following translocation of compound 1. Fig.S9 The structure of compound 4 which was synthesized by linking two P7 molecules through 1,6-bismaleimidohexane. Fig.S10. Detection of the translocation of compound 4 through an α-hl pore from the cis side in the presence of Tris-HCl (ph = 7.98). (a) Current traces of compound 4 at mv. (b) Scatter plot of compound 4 at mv. The events are distributed from i/i 0 = 0.2 to 1.0 at mv. (c) The histogram of blockage currents fits with a Gaussian function at mv. (d) The distributions for the blockage duration are divided into two parts: for times less than the time corresponding to the peak value (t = 0.45 ms), the curve follows a relatively steep rise, and for times greater than the time corresponding to the peak value, the curve is well approximated by an exponential decay, with a fitted value of 1.44 ± 0.16 ms. (d) Current traces of compound 4 at mv. (e) Scatter plot of compound 4 at mv. (f) The distribution of the blockage currents fits with a Gaussian function at mv. There are two populations, one at i/i 0 = 0.25 ± 0.04 and one at i/i 0 = 0.62 ± (f) 11 Histogram of the durations for compound 4 at mv. A Gaussian function was used to fit this distribution, which gave the value of t = 0.33 ± 0.05 ms. 8. Analyzing the disassembly compound 1 and 2 using an α-hl pore Our studies show that a small population of compound 1 and 2 adopt a monomeric-linear structure due to the disassembly of the peptide component in solution. The passage of a molecule through a nanopore is a single molecule event, which provides structural information about each molecule. About 10% of all translocation events of compound 1 and 2 are sorted into the types as partial-then-deep (Fig.S11a) and deep-then-partial (Fig.S11b), illustrating that nanopore biosensor could distinguish compound 1 and 2 with monomeric-linear peptide among all the species. After analyzing these events for compound 1 in great detail, two current levels IV and V were observed. The current distributions of each level were fitted to a Gaussian function (Fig.S12a) which gives a i IV /i 0 = 0.62 ± 0.03 for level IV (i IV ) and i V /i 0 = 0.81 ± 0.02 for level II (i V ). Note that the values of i IV and i V are similar to the blockage current of the monomeric linear form of P7 (Fig.S1b) and of the 20-mer oligonucleotide sequence (Fig.S3b), respectively. Thus it is reasonable to assume that the differences in the observed blockage currents are caused by a gradual entry of the disassembly compound 1 into the β-barrel part of the α-hl pore. The partial-then-deep blockage is responsible for the monomeric P7 entering into pore first and the deep-then-partial blockage is attributed to B20 leading the molecule into the pore. The duration time of these types of events is 0.40 ± 0.04 ms (Fig.S12b), smaller than the events caused by compound 1 that adopts a triple helical collagen-like arrangement. The similar distributions of blockage current were carried out in the nanopore assay of compound 2 (i IV /i 0 = 0.61 ± 0.04, i IV /i 0 = 0.81 ± 0.03), comfirming the reproducibility of these two types of events (Fig.S13a). As a result of the increased length of the oligonucleotide component, the lifetime of partial-then-deep and deep-then-partial events for compound 2 increased to 0.55 ± 0.04 ms (Fig.S13b). Therefore, the α-hl nanopore can be used to discriminate the different structures of compound 1. 12 Fig.S11 Representative nanopore translocation events for compound 1 in which P7 adopts a monomeric linear form. (a) The partial-then-deep current trace (left) and schematic representation (right) accounts for P7 leading the molecule to threading through the pore from cis side. (b) The deep-then-partial current traces (left) and schematic representation (right) illustrates B20 leading the molecule to threading through the pore from cis side. Each event has two current levels, labelled as IV and V. Fig.S12 (a) Distribution of the blockage current collected from individual events of compound 1 in which P7 adopts a monomeric linear form. PIV and PV collected from the current level IV and PV of each event, respectively. The two current distributions, PIV and PV, are fitted into Gaussian function. (b) A single exponential function fits to the duration time histograms. The events were obtained from recordings in 0.1 M compound 1 at ph=7.8 Tris-HCl. 13 Fig.S13 (a) Distribution of the blockage current collected from individual events of compound 2 in which P7 adopts a monomeric linear form. PIV and PV collected from the current level IV and PV of each event, respectively. The two current distributions, PIV and PV, are fitted into Gaussian function with the peak current values of 0.61 and 0.81, respectively. (b) A single exponential functi
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