Enhanced Resolution of Low Molecular Weight Poly(Ethylene Glycol

Dec 2, 2014 - A design with conjugation of DNA hairpin structure to the poly(ethylene glycol) molecule was presented to enhance the temporal resolutio...
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Enhanced Resolution of Low Molecular Weight Poly(Ethylene Glycol) in Nanopore Analysis Chan Cao, Yi-Lun Ying, Zhen Gu, and Yi-Tao Long* Key Laboratory for Advanced Materials & Department of Chemistry, East China University of Science and Technology, Shanghai 200237, P. R. China S Supporting Information *

ABSTRACT: A design with conjugation of DNA hairpin structure to the poly(ethylene glycol) molecule was presented to enhance the temporal resolution of low molecular weight poly(ethylene glycol) in nanopore studies. By the virtue of this design, detection of an individual PEG with molecular weight as low as 140 Da was achieved at the single-molecule level in solution, which provides a novel strategy for characterization of an individual small molecule within a nanopore. Furthermore, we found that the current duration time of poly(ethylene glycol) was scaled with the relative molecular weight, which has a potential application in singlemolecule detection.

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ultrasensitive detection at the single-molecule level in real time.32 However, PEG with low molecular weight traverses through α-HL at an ultrahigh speed, which produces very shortlived blockages. Generally, these short-lived blockages are hard to acquire and recognize due to the low signal-to-noise ratio. Recently, using circuit response theory to model the short-live blockages has allowed the characterization of PEG with molecular weight as low as 370 Da, which requires an additional measurement of the impedance of the single α-HL nanopore together with a high sampling rate of 500 kHz.33 Previous studies in our group showed that the unzipping/ unfolding of secondary structure of ssDNA would generate long-lived characteristic blockages which can be easily recognized.34−36 In order to discriminate low molecular weight PEG, we take advantage of the DNA hairpin structure to generate long-lived characteristic blockages37 (Figure 1a). The conjugation of the DNA hairpin structure to PEG reduces the translocation speed of single unconjugated PEG molecule through an α-HL pore, which ensures a high signal-to-noise ratio. After examining a set of PEG molecules, the identification of single PEG with molecular weight as low as 140 Da was achieved using an α-HL nanopore, and the duration time of current blockage was scaled with the increase of the PEG molecular weight.

oly(ethylene glycol) (PEG) represents a class of molecules, with unique properties such as hydrophilicity, flexibility, and nontoxicity.1 Both high molecular weight and low molecular weight PEG molecules have a wide range of applications in chemical, biological, medical, and clinical areas.2,3 For example, PEG with molar masses from 1 kDa to 5 kDa are often used for the conjugation of drugs, antibodies, and nanoparticles.4,5 Low molecular weight PEG variants 200− 400 Da are widely applied in pharmaceutical products as solvents in oral liquids and soft capsules.6 Even lower molecular weight PEG can be used to treat the skin burns.7 Thus, an analysis of the molecular weight of PEG is very important and essential for applications and controlling the properties of PEG. Although, techniques such as gel electrophoresis8 and mass spectrometry9,10 are available to discriminate the molecular weight of PEG molecules, these techniques could only provide information about the bulk solution rather than the individual features of single PEG molecule. Single-molecule technology breaks the limitations of studies on bulk solution with ensemble averages and uncovers the properties of an individual molecule. α-Hemolysin (α-HL) nanopore was used as a single-molecule analytical method to detect a wide range of analytes including ions,11,12 single stranded DNA (ssDNA) and RNA, 13−17 proteins and peptides,18−24 small organic molecules,25−27 as well as PEG polymer.28−30 Previous studies demonstrated that a PEG molecule could partition into an α-HL nanopore and induce a decrease in ionic conductance. The amplitude of the current blockage depends on the molecular weight of PEG. On account of the long polymer−pore interaction time, α-HL based singlemolecule mass spectrometry achieved to discriminate the molecular weight of PEG between 1100 and 2200 Da.31 Compared with traditional mass spectrometry, nanopore singlemolecule mass spectrometry is free of ionization and has an © 2014 American Chemical Society

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EXPERIMENTAL SECTION Reagents and Chemicals. α-HL, decane (anhydrous, ≥99%), and polydisperse PEG (pPEG) (molecular weight, 190−210) were purchased from Sigma-Aldrich (St. Louis, Received: November 12, 2014 Accepted: December 2, 2014 Published: December 2, 2014 11946

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Letter

RESULTS AND DISCUSSION In our experiments, a series of DNA-PEG-DNA conjugates (DPD) were designed (Figure 1a). A PEG molecule was conjugated to two oligonucleotide strands (Oligo1 and Oligo2) at each end of the oligomers. Oligo1 acted as a guide to lead PEG molecule to enter the vestibule of α-HL, forming a fourbase pair DNA structure with the Oligo2 (Figure S1 in the Supporting Information). The hairpin structure in DPD aims to slow down the translocation speed of the PEG molecule and enhance the temporal resolution of PEG. When setting the filter of the amplifier at 3 kHz and a sampling rate at 100 kHz, the amplifier hardly acquired any current blockages after adding 1 μM pPEG into the cis side of α-HL at a potential of 120 mV. This phenomenon was retained even at the high concentration of 1 mM (Figure S2 in the Supporting Information). As a first step to verify the design of DPD, the DPD-3 containing PEG140 was added to the cis side of α-HL at an applied potential of +120 mV. As depicted in Figure 1b, the translocation of DPD-3 from cis to trans side of α-HL generated characteristic three-step current blockages which contain Level1, Level-2, and Level-3. Here, I0 was defined as the open pore current and I as the blockage current when analyte stayed within the pore. Hence, the ratios of three current levels are assigned as I1/I0, I2/I0, and I3/I0, respectively. As shown in Figure 2a,b, the Level-1 had an I1/I0 of 0.68 ± 0.01 with a

Figure 1. (a) Upper: illustration of a DNA-PEG-DNA (DPD) traversing through the α-HL pore. A biological α-HL nanopore was embedded in a lipid bilayer. The two compartments of the bilayer cell are termed cis and trans. The potential was applied from the trans side using a pair of Ag/AgCl electrodes. The current traces were recorded in solutions containing 1.0 M KCl, 10 mM Tris, and 1 mM EDTA buffer (pH = 7.98) at +120 mV. Bottom: molecular structure of DPD. A PEG molecule conjugates with two single strand oligonucleotides which may form a four-base pair DNA hairpin structure. (b) Upper: the raw data of current blockages in the presence of 1 μM DPD molecule in the cis compartment. Red arrows indicate the typical threestep blockages. Bottom: a typical three-step current blockage for the translocation of the DPD molecule through an α-HL nanopore. The three current levels are labeled as Level-1, Level-2, and Level-3, respectively.

MO). 1,2-Diphytanoyl-sn-glycero-3-phosphocholine (chloroform, ≥99%) was purchased from Avanti Polar Lipids Inc. (Alabaster, AL). All DPD conjugates, DNA-1 (5′-GTC ACG ATG GCC CAG TAG TTA AAA ATT GAT GAC CCG GTA GCA CTG-3′) and DNA-2 (5′-AAA AAA AAA AAA AAA AAA AA-PEG3-AA AAA AAA AAA AAA AAA AAA-3′) were synthesized and HPLC-purified by Sangon Biotech (Shanghai) Co. Ltd. (China). All reagents and materials are of analytical grade, and solvents were purified by standard procedures. All solutions for analytical studies were prepared with ultrapure water (reaching a resistivity of 18.2 MΩ cm at 25 °C) obtained using a Milli-Q System (EMD Millipore, Billerica, MA). Nanopore Electrical Recording. The nanopore detection method was conducted according to our previous studies.26 The lipid bilayer membrane was formed spanning a 50 μm orifice in the center of a Delrin bilayer cup (Warner Instruments, Hamden, CT) that partitioned into two chambers, which are termed cis and trans. The stability of the bilayer was determined by monitoring its resistance and capacitance. The solutions on each side of the two chambers containing 1.0 M KCl were buffered with 10 mM Tris and 1 mM EDTA (pH = 7.98). The α-HL was injected to the aperture in the cis chamber, and the pore insertion was determined by a welldefined jump in the current value. The DPD, DNA-1, and DNA-2 were added to the cis solution. The cis compartment was defined as the virtual ground and the voltage was given from the trans. Thus, negatively charged DNA could be driven from the cis to the trans side at a positive potential. The current trace was amplified and measured via a ChemClamp (Dagan Corporation, Minneapolis, MN) instrument with a 3 kHz lowpass Bessel filter. Data were acquired at a sampling rate of 100 kHz by using a DigiData 1440A converter and a PC running PClamp 10.3 (Axon Instruments, Forest City, CA). Data analysis was performed using a home-designed software (http://people.bath.ac.uk/yl505/nanoporeanalysis.html) 38 and OriginLab 8.0 (OriginLab Corporation, Northampton, MA). Nanopore measurements were conducted at 24 ± 2 °C.

Figure 2. (a) Histograms of blockage currents for Level-1, Level-2, and Level-3 (from top to bottom), respectively. Each histogram was fit to Gaussian distributions. (b) Histograms of duration time for Level-1, Level-2, and Level-3 (from top to bottom), respectively. The histogram of Level-1 was fit to an exponential function. A Gaussian function was used to fit the duration time of both Level-2 and Level-3, respectively. (c) Illustration of the translocation process of the DPD molecule transport through the α-HL nanopore.

duration of 0.42 ± 0.01 ms. Previous studies demonstrated that the four-base pair hairpin structure induced the I/I0 around 0.6.39 Thus, the blockage current of Level-1 indicated that the leading part of the 20-base oligonucleotide guided the hairpin structure together with the PEG molecule to enter the vestibule of the α-HL. As shown in the research of ssDNA with a nanopore, the duration time of poly(dC)20 traversing through an α-HL is 0.02 ms at +120 mV,40 which is almost 20 times 11947

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nanopore (Figures S5−S7 in the Supporting Information). PEG-6, 9, 12 corresponds to the PEG molecular weight of 260, 400, and 530 Da, respectively. As illustrated in Figure 3a, the

shorter than that of Level-1. Thus, the unzipping process of the hairpin structure produced the duration time of Level-1. Then, the blockage current of DPD-3 rose to Level-2 with an I2/I0 of 0.50 ± 0.01 that lasted for a comparably short time of 0.21 ± 0.01 ms. This finding agrees with previous studies of the interactions between PEG and α-HL that the PEG molecule formed by repeat monomer units less than 20 (molecular weight < 880) generated the lower amplitude of current blockage (I/I0 < 0.5).41 As for DPD-3, the value of I2/I0 suggested the translocation of the PEG part. Eventually, the current of three-step blockage dropped to the Level-3 displayed a nearly full blockage of the open pore current. The statistical analysis showed that the value of I3/I0 is 0.83 ± 0.01 and the duration time of Level-3 is 0.22 ± 0.01 ms. The current amplitude and duration of Level-3 are consistent with previous studies of ssDNA translocation through the a-HL nanopore,42 which illustrates that Level-3 is assigned to the translocation of the linear form of DPD. In order to confirm that Level-2 originated from the translocation of the PEG part, a control DNA-1 containing four-base hairpin structure was designed and analyzed by an αHL nanopore (Figure S3 in the Supporting Information). Compared with the DPD-3, 5-mer polydeoxyadenosines was used to replace the PEG-3 part in the design of the DNA-1. Different from the DPD-3, the typical three-step level blockages were not observed in the nanopore experiments of DNA-1. The translocation events of DNA-1 are shaped like a “shoulderspike” (Figure S3 in the Supporting Information). This twostep signal is accordance with the studies of DNA hairpin molecules traversing through the α-HL nanopore.39 It should be noted that the control DNA-1 could not induce the decrease in blockage current as Level-2 of DPD-3. Therefore, the PEG-3 part of the DPD-3 molecule should be responsible for the transient blockage of Level-2 in three-step signals. To further interpret this phenomena, a control DNA-2 was incorporated by replacing Oligo 1 and Oligo 2 with two 20-mer polydeoxyadenines, respectively. Obviously, the DNA-2 could not generate the hairpin structure. The translocation events of the control DNA-2 are shaped like a spike (Figure S4 in the Supporting Information), which is neither like the characteristic three-step current blockages produced by DPD-3 nor “shoulder-spike” current that the control DNA-1 performed. The duration time of the control DNA-2 is 0.25 ± 0.01 ms, which is almost 4 times faster than that of DPD-3. These results further confirmed that the hairpin structure efficiently slowed down the translocation speed of low molecular weight PEG. The capture of hairpin structure was responsible for the current of Level-1 while the translocation of unzipped hairpin induced the current value of Level-3. Therefore, the three-step blockages of DPD are appointed to the three sequential steps (Figure 2c): First, DPD was captured in the vestibule of α-HL and underwent an unzipping process. Second, the PEG part of DPD translocated the narrowest construction of the α-HL after completing the unzipping process of the hairpin structure, resulting in a clear current step as Level-2. Third, the linear form of DPD rapidly transported through the α-HL, leading to a deep and rapid current blockage of Level-3. This mechanism showed that our design of the DPD molecule could enhance the temporal resolution of low molecular weight PEG. The characteristic three-step blockages were easily identified among a variety of blockages including bumping events. In the next step, a series of DPD molecules with increasing PEG repeat units of n = 6, 9, 12 were analyzed via an α-HL

Figure 3. (a) Typical translocation events of DPD-3 (orange), DPD-6 (green), DPD-9 (blue), and DPD-12 (black), respectively. (b) Histograms of duration time for the Level-2 current blockages caused by DPD-3 (orange), DPD-6 (green), DPD-9 (blue), and DPD-12 (gray), respectively. (c) The duration time of Level-2 for PEG-3, PEG6, PEG-9, and PEG-12, respectively.

current traces of DPD-6, DPD-9, and DPD-12 are also shaped in three-steps as that of DPD-3. The duration of Level-2 increased from 0.21 ± 0.01 ms to 0.35 ± 0.01 ms as the molecular weight of PEG increased from 140 to 530 Da (Figure 3b). Therefore, the characteristic duration time scaled with the molecular weight of the PEG polymer and further confirms that the Level-2 is caused by the PEG part. Our DPD design exhibits excellent discrimination of low molecular weight PEG, even down to as few as three monomers. Besides, the current and duration values of Level-1 and Level-3 are almost kept constant with the increase of molecular weight of PEG fragment (Table 1). These results further demonstrated that the increasing molecular weight of PEG did not change the translocation mechanism of DPD conjugates, which ensures the clear temporal resolution of PEG. 11948

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Table 1. Blockage Currents and Durations of the Three-Level Signalsa analyte DPD-3 DPD-6 DPD-9 DPD-12

I1/I0 0.68 0.64 0.64 0.62

± ± ± ±

0.01 0.01 0.01 0.02

I2/I0 0.50 0.50 0.49 0.49

± ± ± ±

0.01 0.01 0.01 0.01

I3/I0 0.83 0.82 0.79 0.82

± ± ± ±

t1 (ms)

0.01 0.01 0.02 0.01

0.42 0.40 0.43 0.42

± ± ± ±

t2 (ms)

0.01 0.01 0.01 0.01

0.21 0.26 0.28 0.35

± ± ± ±

0.01 0.01 0.02 0.01

t3 (ms) 0.22 0.22 0.20 0.26

± ± ± ±

0.01 0.01 0.01 0.02

a

The histograms of blockage currents and blockage durations for DPD-6, DPD-9, and DPD-12 are shown in the Supporting Information. Data values were based on three separate experiments.

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CONCLUSION In summary, we demonstrated that a DPD molecule design could discriminate low molecular weight PEG at the singlemolecule level. A four-base hairpin structure was introduced in this design to enhance the temporal resolution of the short PEG molecule whose nanopore blockages are barely acquired by a traditional current amplifier. Our results reveal that the DPD molecule produces an easily recognized three-level blockage. The duration time of Level-2 scales with the molecular weight of PEG. By analyzing Level-2 duration, discrimination of the molecular weight of PEG as low as 140 Da was achieved. Moreover, this DPD molecular design provides a novel strategy for characterization of a single small molecule which is otherwise hard to detect due to rapid translocation through the nanopore.

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ASSOCIATED CONTENT

S Supporting Information *

Ultraviolet melting curves of DPD compounds (Figure S1), detection of pPEG using α-HL pore (Figure S2), analyzing the control DNA-1 using α-HL pore (Figure S3), analyzing the control DNA-2 using α-HL pore (Figure S4), detection of DPD-6 using α-HL pore (Figure S5), detection of DPD-9 using α-HL pore (Figure S6), and detection of DPD-12 using α-HL pore (Figure S7). This material is available free of charge via the Internet at http://pubs.acs.org.

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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 Science Fund of China (21327807) and National Base Research 973 Program (2013CB733700). Y. T. Long. is supported by the National Science Fund for Distinguished Young Scholars of China (21125522).

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REFERENCES

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