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Biomaterials

journal homepage: www.elsevier.com/locate/biomaterials

Nano-channel of viral DNA packaging motor as single pore to differentiatepeptides with single amino acid difference

Zhouxiang Ji, Xinqi Kang, Shaoying Wang1, Peixuan Guo∗

Center for RNA Nanobiotechnology and Nanomedicine, Division of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, College of Medicine, Dorothy M.Davis Heart and Lung Research Institute and James Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA

A R T I C L E I N F O

Keywords:NanoporeT7 DNA packaging motorProtein sequencingSingle pore sensingBiomotorBacteriophage DNA packaging

A B S T R A C T

Detection, differentiation, mapping, and sequencing of proteins are important in proteomics for the assessmentof cell development such as protein methylation or phosphorylation as well as the diagnosis of diseases includingmetabolic disorder, mental illness, immunological ailments, and malignant cancers. Nanopore technology hasdemonstrated the potential for the sequencing or sensing of DNA, RNA, chemicals, or other macromolecules. Dueto the diversity of protein in shape, structure and charge and the composition versatility of 20 amino acids, thesequencing of proteins remains challenging. Herein, we report the application of the channel of bacteriophageT7 DNA packaging motor for the differentiation of an assortment of peptides of a single amino acid difference.Explicit fingerprints or signatures were obtained based on current blockage and dwell time of individual peptide.Data from the clear mapping of small proteins after protease digestion suggests the potential of using T7 motorchannel for proteomics including protein sequencing.

1. Introduction

Proteomics, a challenging but highly significant field, is imperativein understanding the biological function and role of certain proteins inthe onset of various genetic diseases, immunological aberrations, me-tabolic disorders, and cancer ailments [1–3]. In its current stage, thefingerprinting of polypeptides or proteins is mainly dependent on massspectrometry (MS), which currently has not reached the sensitivity ofsingle molecules [4,5]. In order to advance the field of proteomics,nanopore technology provides an alternative approach for peptidefingerprinting at a single molecule level. The principle of nanoporedetection is based on the resistive pulse technique, that is, analytes aredriven through a pore by electric force to produce a profile of pico-ampere scale current signatures characterizing each analyte in interest[6–8].

Currently, nanopore technology primarily focuses on nucleic acidcharacterization [9–13]. A few studies have shown great potential forpeptide or protein sensing [14–18]. For example, the protein size,fluctuation, and conformational changes have been investigated bysolid-state nanopore [19–22]. In a particular study α-hemolysin wasused in real-time monitoring of peptide cleavage [23,24]. Other studiesutilizing other portals, such as Cly A for protein folding, [25,26] Phi29

connector for the distinguish of peptides, [27] and SPP1 connector forpeptide oligomerization [28,29] have also been reported.

Biological motors are very common in living systems, facilitatingvarious functions [30–36]. In dsDNA animal or bacterial viruses such asPhi29, SPP1, T3, T4, T5, and T7, their genome enters and exits thebacteriophage capsid during replication and infection, respectively,through a portal protein channel called the connector [37–43]. Bac-teriophage T7 belongs to the Podoviridae family, which infects Escher-ichia coli (E.coli). Data from cryo-electron microscopy reveals that theT7 connector is composed of twelve subunits encoded by gene 8 (gp8)with a molecular weight of 59 k Da [44,45]. In this study, the T7connector gene was cloned and expressed in E. coli. The purified T7connector was inserted into a lipid bilayer membrane as a biosensor forthe fingerprinting of different peptides. Their blockage or dwell timewas then used as an explicit signature of each peptide. Clear mappingsof several peptides were achieved after digestion with protease, sug-gesting the potential of using T7 nanopore technology for proteomicsand protein sequencing.

https://doi.org/10.1016/j.biomaterials.2018.08.005Received 11 June 2018; Received in revised form 17 July 2018; Accepted 2 August 2018

∗ Corresponding author. Sylvan G. Frank Endowed Chair in Pharmaceutics and Drug Delivery, Director of Center for Nanobiotechnology and Nanomedicine, TheOhio State University, 460 W. 12th Avenue, Biomedical Research Tower, Rm. 912, Columbus, OH, 43210, USA.

1 Current address : P&Z Biological Technology, Newark, NJ, USA.E-mail address: guo.1091@osu.edu (P. Guo).

Biomaterials 182 (2018) 227–233

Available online 03 August 20180142-9612/ © 2018 Elsevier Ltd. All rights reserved.

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2. Material and methods

2.1. Materials

Adenosine triphosphate (ATP) was obtained from VWR. Imidazolewas purchased from Acros. 1, 2-diphytanoyl-sn-glycero-3-phosphocho-line (DPhPC) was purchased from Avanti Polar Lipids. The peptides inthis study are synthesized by Genescript. The trypsin was purchasedfrom Sigma and dissolved in dd H2O. Other chemicals were from FisherScientific, if not specified. The sequences of peptides in this study are:

(1) R8: RRRRRRRR;(2) R9: RRRRRRRRR;(3) R10: RRRRRRRRRR;(4) R11: RRRRRRRRRRR;(5) R12: RRRRRRRRRRRR

2.2. Cloning, expression and purification of T7 connector

The plasmid to express T7 connector was synthesized and con-structed by Genescript. The gene 8 (gp8) encoding single subunit of T7connector with the deletion of ADSVGLQPGI in the C-terminal, and theDNA fragment encoding six histidine in the C-terminal, were insertedbetween NdeI and BamHI of the pET-3c vector [46,47]. The newly-constructed plasmid was transformed to E. coli HMS 174 (DE3). Theexpression and purification procedure are similar to phi29 connector[10,27,48]. The E. coli containing the plasmid for T7 expression wascultured in 500ml standard Luria broth (LB) medium at 37 °C untilOD600 reached 0.5–0.6. The T7 protein expression was induced with theaddition of IPTG (final concentration: 0.5 mM). The cultures weregrown for additional 3 h before harvest by centrifugation (5000 rpm for30min). The pellets were resuspended in buffer (Tris-HCl 0.1M, NaCl0.5 M, ATP 10mM, Glycerol 14.4%, Imidazole 5mM) and then cellswere lysed by French Press. The soluble protein was recovered bycentrifugation (11000 rpm for 30min) and supernatant was passedthrough a 0.45 μm membrane filter before loading to purification

column (Thermo Fisher Scientific) filled by His•Bind® resin (EMD Mil-lipore). The T7 connector protein was eluted using elution buffer (Tris-HCl 0.1 M, NaCl 0.5 M, ATP 50mM, Glycerol 14.4%, Imidazole 1M).The purified T7 protein was analyzed by 12% SDS-PAGE.

2.3. Incorporation of T7 connector into liposomes

The incorporation process contains dehydration and hydration steps[10,48,49]. Briefly, 100 μl 10 mg/ml DPhPC in chloroform was driedunder vacuum by the evaporator (Buchi). The T7 connector (finalconcentration 100–500 μg/ml) and liposome buffer (Liposome buffer:3M KCl, 250mM sucrose, 5 mM HEPES, pH 7.4) were added and vor-texed thoroughly. Then the mixture was filtered through 0.4 μm poly-carbonate membrane for around 30 times using the extruder (AvantiPolar Lipids) in order to produce homogenous proteoliposomes.

2.4. Electrophysiological assays

The lipid bilayer membrane was formed on the Teflon partitionmembrane (pore size: 200 μm) which separates the whole chamber intotwo compartments, cis- and trans-chamber [48]. A pair of Ag/AgClelectrodes were placed in both chambers. Bilayer Clamp Amplifier BC-535 (Warner Instruments) or Axon 200B (Molecular Devices) wasconnected to the Axon DigiData 1440 A analog-digital converter (Mo-lecular Devices). Data was recorded at 1 K Hz bandwidth with a sam-pling frequency 20 KHz. The Clampex 10 (Molecular Devices), Clampfit10 (Molecular Devices), MOSAIC [50] and Origin 8 (Origin Lab Cor-poration) were used to collect and analyze data. The events thatlast< 0.4 ms were excluded for translocation data analysis.

2.5. Peptide translocation assays

The peptide was added into chambers after single channel insertionor peptide was premixed with conductance buffer (0.15M KCl, 5 mMHEPES, pH 7.4) for the detection of single type of peptide (63 nM). Themixture of R8, R9, R10 and R12 peptides was kept the same

Fig. 1. Overview of bacteriophage T7 connector. (a–b) The top view (a) and side view (b) of T7 connector (PDB:3J4A), showing the dimension, the positivelycharged (blue), negatively charged (red), and neutral (white) amino acids. (c) 12% SDS-PAGE showing the purified C-His tagged T7 connector (lane 2, 59 k Da)overexpressed in E. coli. Lane 1: molecular weight marker; lane 3: phi29 connector (37 k Da) as a control. (d) Current trace of single T7 connector insertion withpolarity voltage switch of 50mV. (e) Histogram of conductance of T7 connector based on 117 insertion events. Buffer: 0.15M KCl, 5 mM HEPES, pH 7.4.

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concentration (21 nM) in buffer.

2.6. Peptide cleavage assay

The R11 (RRR RRR RRR RR) or R12 (RRR RRR RRR RRR) waspremixed with conductance buffer. After single connector was insertedand then the translocation signals of R11 or R12 peptide were observedunder −50mV, the trypsin was added and mixed into the cis-chamber,while recording the data until the majority of R11 or R12 was digested[51].

3. Results and discussion

3.1. Cloning and expressing the T7 connector in E. coli and insertion of thepurified connector into lipid bilayer membrane

The gene gp8, which codes for the connector protein of the bac-teriophage T7 DNA packaging motor was cloned into the plasmid pET-3c through the fusion of a six-aa His-tag into the C-terminus, facilitatingprotein purification [46,52]. After induction by IPTG, the over-expressed gp8 gene production was spontaneously assembled into its

Fig. 2. Differentiation of peptides with 8, 9, 10, 11, 12 arginine (R) residues. The blockage is defined as (Io-Ib)/Io× 100% in which Ib is the block current duringtranslocation and Io is the current of open channel without translocation, and fitted by a Gaussian distribution. Peptide concentration: 63 nM. Applied voltage:−50mV. Buffer: 0.15M KCl, 5mM HEPES, pH 7.4.

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channel complex (Fig. 1a and b). The connector was purified intohomogeneity based on 12% SDS-PAGE result (Fig. 1c). Unlike othermembrane proteins, viral motor channels cannot be inserted into a lipidbilayer membrane without mediating vesicle liposome or porphyrin[10,53]. For this reason, the purified T7 connector was incorporatedinto the liposome to form the proteoliposome which was then insertedinto a lipid bilayer membrane by fusion as reported previously [10,48].Single channel characterization (Fig. 1d) revealed that the conductanceof T7 connector was 0.73 ± 0.10 nS, based on the measurement of 117channels under 50mV (Fig. 1e). Cloning and expression of the T7connector gene and subsequent connector insertion in the lipid bilayermembrane resulted in an assembled nanopore that will ultimately beutilized for characterization and mapping of peptides.

3.2. Differentiation of peptides of varying residues by current blockage

Considering the principle of nanopore sensing, we first investigatedfive different lengths of arginine peptides consisting of 8 (R8; Fig. 2a, b,c), 9 (R9; Fig. 2d, e, f), 10 (R10; Fig. 2g, h, i), 11 (R11; Fig. 2j, k, l), and

12 (R12; Fig. 2 m, n, o) residues, which are positively charged. Theblockage and dwell time were used to characterize the peptides. Theblockage percentage was calculated using the equation (Io-Ib)/Io× 100%, in which Io is the channel current in the absence of peptidetranslocation (open channel current), and Ib is the current duringpeptide translocation (block current). Presently, we only focus on thedifferentiation of peptides based on blockages, which were fit byGaussian distributions. The blockages of R8, R9, R10, R11, R12 were44.58% ± 1.74%, 50.70% ± 0.93%, 54.61% ± 0.81%,57.77% ± 1.20%, 60.55% ± 0.77%, respectively (Fig. 2b, e, h, k, n).Each blockage peak for each peptide had relatively good separation, atleast ∼3%. The electrical signal resulting from the peptide passingthrough the channel is related to the length of the narrow region of thechannel. Unsurprisingly, longer peptides have larger blockage percen-tages and longer dwell time. This indicates that the length of peptideswith twelve amino acids is shorter than the length of the narrow regionof T7 connector and each amino acid contributes to the blockage anddwell time. Peptides were driven to translocate through T7 connectorby electric force, and the differences of amino acids in mass, charge and

Fig. 3. Discrimination of R8, R9, R10 and R12 peptides in a mixture. (a-c) The electric profiles of the mixture were shown in the blockage histogram (a), currenttrace (b) and scatter plot (c). R represents arginine. Different colors represent different peptides. The histograms of blockages were fitted by four Gaussian dis-tributions. Applied voltage: −50mV. Concentration of each peptide in the mixture is 21 nM. Buffer: 0.15M KCl, 5mM HEPES, pH 7.4. More than 10,000 events wereanalyzed in the histogram or scatter plot.

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Fig. 4. Mapping of the 11-aa and 12-aa peptides cleaved by trypsin. (a–f) The electric profiles of R12 before (a–c) and after (d–f) addition of trypsin. Beforetrypsin digression, only one peak of 60.57% ± 0.45% blockage was found for R12. With the addition of trypsin (final concentration: 25 ng/ml) into the cis-chamber,multiple peaks that represent the cleaved products were shown in the trace (d), the blockage histogram (e), and the scatter plot (f). The blockage levels of45.29% ± 0.82%, 51.23% ± 0.54%, and 54.91% ± 0.48% represent R8, R9 and R10, respectively. The blockage level of 60.24% ± 0.42% matches the R12blockage level before adding trypsin. (g–l) The electric profiles of R11 before (g–i) and after (j–l) addition of trypsin. The trace (g), blockage histogram (h), scatterplot (i) of R11 showed single blockage peak of 57.68% ± 1.22%. Two additional peaks of 45.06% ± 1.38% and 51.59% ± 2.27% were generated after addition oftrypsin (final concentration: 50 ng/ml), matching the blockage level of R8 and R9. Arrows indicate the cleavage sites of each R11 and R12 by trypsin. The histogramsof blockages were fitted by single or multiple Gaussian distributions. e and f were recorded 10min after the addition of trypsin, and k and I were recorded 8min afteradding trypsin. The recording time for the plotted data (b, c, e, f, h, i, k, l) was 5min. The R11 or R12 peptide concentration: 63 nM. Applied voltage:−50mV. Buffer:0.15 M KCl, 5mM HEPES, pH 7.4.

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size could affect current blockage and dwell time in principle.

3.3. Discriminating peptides of varying size in mixture

The T7 motor channel was investigated for its potential to differ-entiate peptides in mixtures of biological samples. As shown in Fig. 2,the blockage peaks for each peptide showed adequate separation. TheR8, R9, R10, and R12 peptides were kept at a constant concentration(21 nM) for the nanopore translocation experiment (Fig. 3). FromFig. 3a and b, there were four distinct peaks of blockages at45.20% ± 1.55%, 50.80% ± 0.96%, 54.65% ± 0.75, and59.89% ± 0.64% as a result of fitting the data by Gaussian distribu-tions. These peaks matched the blockage parameters of individualpeptides measured separately. Although current blockage has beenextensively used as a reliable and sensitive signature for a variety ofanalytes, dwell time parameters have been reported to be hetero-geneous with large variation [54–56]. Surprisingly, clear and distinctdwell time signatures were obtained with discrete order when R8, R9,R10, and R12 peptides were present in a mixture (Fig. 3c).

3.4. Mapping of 11-aa and 12-aa peptides by real-time sensing via trypsincleavage

In the interest of assessing the potential of the T7 channel for pro-tein mapping, two peptides were digested by trypsin. R11 or R12peptides (63 nM) were premixed with buffer in chambers, and trans-location signals were recorded after single channel insertion.Subsequently, trypsin was added into the cis-chamber and mixed whiletranslocation events were continuously recorded. As shown in Fig. 4a,b, c, the blockage of R12 was 60.57% ± 0.45%. After the addition oftrypsin, three new peaks appeared, 45.29% ± 0.82%,51.23% ± 0.54%, and 54.91% ± 0.48%; these results suggested thatpeptide R12 was cut into multiple fragments, R8, R9, and R10 (Fig. 4d,e, f). However, peptides composed of less than 8 amino acids were notdetectable by T7 connector.

Trypsin is considered to be an endopeptidase, which means that itdoes not digest arginine at the C-terminal. Mapping of R12 via proteasedigestion did not reveal the blockage peak corresponding to R11(Fig. 4e). To confirm that the missed peak of R11 was not due to theissue of overlap of the blockage peaks between R11 and R12, R11cleavage was carried out in parallel. Before adding trypsin to thechamber, there was a single blockage peak of 57.68% ± 1.22% re-presenting R11 (Fig. 4 g, h, i); however, with the addition of trypsin tothe cis-chamber, the translocation events corresponding to R11 de-creased with time, while two new peaks with 45.06% ± 1.38% and51.59% ± 2.27%, respectively, appeared (Fig. 4j, k, l). These twopeaks matched the two peaks representing R8 and R9 in Fig. 2. Therewas no R10 blockage peak in R11 digestion by trypsin. The peptidemapping of R11 and R12 support a notion that trypsin is an en-dopeptidase and at least two amino acids at the C-terminal are requiredfor cleaving.

A variety of protease assays have been used for the diagnosis ofdifferent diseases [57–59]. For example, trypsin has been used as themost reliable marker for the diagnosis of pancreatitis. Therefore, thepeptide mapping methods reported here could also be used to monitorserum enzyme activity or diagnosis of various diseases utilizing pro-teases as markers. The results from this study suggest that nanoporetechnology has a potential for diseases diagnosis, in addition to proteinsequencing.

4. Conclusions

Reengineered T7 connector was used as a single pore sensing in-strument to discriminate peptides with a difference of one amino acidresidue. Individual signatures of current variation and dwell time offour peptides were clearly distinguished through use of the nanopore.

The mapping of proteins based on the fingerprints of small peptidesafter trypsin digestion reveals the potential to detect serum proteaseabnormality in diseases diagnosis. In furtherance of the field of pro-teomics, our results pave the way to protein mapping and sequencingusing nanopore technology, and demonstrate additional potential ofprotein pore in biomedical application.

Author contributions

P.G. and Z. J. conceived and designed the experiments in this study;Z. J. and X. K. conducted the experiments; Z. J. analyzed data; S. W.designed cloning; Z. J. and P. G. wrote the manuscript.

Competing financial interests

P.G.'s Sylvan G. Frank Endowed Chair position in Pharmaceuticsand Drug Delivery is funded by the CM Chen Foundation. PG is theconsultant of Oxford Nanopore Technologies, as well as the cofounderof Shenzhen P&Z Bio-medical Co. Ltd, its subsidiary US P&Z BiologicalTechnology LLC, and ExonanoRNA LLC.

Data availability statement

The data that supports the findings of this study are available fromthe corresponding author upon reasonable request.

Acknowledgements

We would like to thank D. W. Binzel, C. Ghimire and D. Driver tomodify manuscript. The research was supported by NIH grantR01EB012135.

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