Nonfunctionalized PNAs as Beacons for Nucleic Acid Detection in a

Letter Cite This: ACS Sens. XXXX, XXX, XXX−XXX

pubs.acs.org/acssensors

Nonfunctionalized PNAs as Beacons for Nucleic Acid Detection in a Nanopore System Alina Asandei,†,# Loredana Mereuta,‡,# Jonggwan Park,§ Chang Ho Seo,§ Yoonkyung Park,*,∥ and Tudor Luchian*,‡ Interdisciplinary Research Institute, Sciences Department and ‡Department of Physics, ‘Alexandru I. Cuza’ University, Iasi, Romania, 700506 § Department of Bioinformatics, Kongju National University, Kongju, South Korea, 32588 ∥ Department of Department of Biomedical Science and Research Center for Proteinaceous Materials (RCPM), Chosun University, Gwangju, South Korea, 61452 Downloaded by UNIV AUTONOMA DE COAHUILA at 19:21:26:458 on May 28, 2019 from https://pubs.acs.org/doi/10.1021/acssensors.9b00553.

†

S Supporting Information *

ABSTRACT: In this work, single-channel current recordings were used to selectively detect individual ssDNA strands in the vestibule of the αhemolysin (α-HL) protein nanopore. The sensing mechanism was based on the detection of the intrinsic topological change of target ssDNA molecules after the hybridization with complementary PNA fragments. The readily distinguishable current signatures of PNA-DNA duplexes reversible association with the α-HL’s vestibule, in terms of blockade amplitudes and kinetic features, allows specific detection of nucleic acid hybridization.

KEYWORDS: protein nanopore, short ssDNA sensing, PNA, single nucleotide substitution, electrophysiology s envisioned by Kasianowicz et al. on the α-hemolysin (αHL) protein nanopore of Staphylococcus aureus,1 various protein-based nanopores have emerged as powerful technologies to provide real-time, label-free nucleic acids polymer sequencing,2−10 despite bottlenecks related to the spatial resolution and accurate reading of high velocity-translocating nucleotide polymers.11,12 There are nonetheless applications requiring information on key genomic subsets. For such applications, sequencing by hybridization was conceived as a method of choice.13−17 This approach is beneficial for validation of small genomic insertion and deletion, haplotype phasing, or in applications requiring detection of DNA fragments which serve as biomarkers and forensic identification.18−22 For such tasks, traditional approaches include melting profile analysis of double-stranded DNAs denaturation,23 high resolution melting techniques,24 microarrays,25 and PCRbased sequencing techniques.26−28 Drawbacks associated with many of these methods include long analysis time (∼ hours), inherent technical limitations, or increased cost. Single-molecule kinetics analysis revealed the potential of nanopores to assay short oligonucleotide hybridization kinetics and single-nucleotide polymorphism detection.29−44 Recently, we used wild type α-HL nanopores in conjunction with polycationic peptide-functionalized peptide nucleic acids (PNAs) to demonstrate indirect detection of ssDNAs.45 The choice of PNAs lies in that, with structural DNA analogues

A

© XXXX American Chemical Society

containing an uncharged N-(2-aminoethyl)-glycine-based pseudopeptide backbone, PNAs mimic ssDNAs in forming Watson−Crick complementary duplexes, and display a greater binding affinity, selectivity, and resistance to degradation by nucleases and proteases.46,47 Notably, for the same degree of complementarity between strands, the neutral character of the PNA results in a stronger stability of PNA-DNA as compared to DNA-DNA duplexes, which is maintained at low to medium ionic strength,46 and the melting temperature of PNA-DNA duplexes is largely independent of salt concentration. Based on nontagged electrically neutral PNAs, we demonstrate here a simpler assay that can directly detect the presence of short ssDNAs, by monitoring the specific signature of the electrophoretically driven confinement of single anionic PNA-DNA duplexes inside the α-HL’s vestibule. Electrophysiology recordings were used to examine the single-channel properties of the α-HL when interacting with various ssDNAs added to the cis chamber (0.5 μM) (Figure 1). As visible, the single-channel currents show reversible changes reflecting the transient blockade of the nanopore by the cis-totrans passage of individual cDNA fragments (Figure 1, II a). Excess addition of the complementary PNA (5 μM) to the cis Received: March 21, 2019 Accepted: May 23, 2019 Published: May 23, 2019 A

DOI: 10.1021/acssensors.9b00553 ACS Sens. XXXX, XXX, XXX−XXX

Letter

ACS Sensors

Figure 1. Illustration of blockade events through the open α-HL induced by ssDNAs or PNA-DNA duplexes with variable mismatch. Typical single-molecule electrical recordings performed at ΔV = +200 mV, with symmetrical salt solutions containing 3 M KCl buffered at pH = 7.2 with HEPES (10 mM) (I, a), reveal that cis-side added cDNA (II, a), nDNA7_5 (II, c), or nDNA1_5 (II, e) at a bulk concentration of 0.5 μM, are readily captured at the vestibule entrance of the nanopore. Individual binding events are seen as negative current deflections. Subsequent cis-side addition of PNA (5 μM) (I, b) leads to the formation of hybridized PNA-cDNA (II, b), PNA-nDNA7_5 (II, d), or PNA-nDNA1_5 (II, f) complexes, whose electrophoretic capture inside the nanopore’s vestibule entails distinct alterations in the dynamics of blockades through the nanopore, depending on the degree of nucleotide mismatch between the strands. The boxes in II, d and II, f, highlight the nucleotide mismatched regions.

chamber, already containing the cDNAs, resulted in (i) the decrease in the frequency of transient blockade events, and (ii) the appearance of a new blockade substate (Figure 1, II b). As discussed previously,45 these phenomena reflect detection of the cis-side hybridized PNA-cDNA duplexes. Addition of ssDNAs presenting seven mismatched bases (nDNA7_5) to the PNA constructs left the blockade amplitude and kinetic features characterizing the association of ssDNA strands to the nanopore largely unchanged (Figure 1, II d), as compared to the instance when only nDNA7_5 was present in the recording chamber (Figure 1, II c). We concluded that the mismatch on the PNA-nDNA7_5 dimer imparted a sizable impact on the duplex stability, precluding its successful capture by the α-HL. This is in line with previous findings, suggesting that complementary sequence recognition and subsequent formation of stable duplexes from short

ssDNA strands are sensitive to the number of complementary base pairs.48 The cis-side hybridization of the PNA with ssDNA containing a single nucleotide mismatch (nDNA1_5) resulted in a particular pattern of current blockades through a single αHL nanopore (Figure 1, II f), distinct from the case when only nDNA1_5 fragments interacted with the nanopore (Figure 1, II e). It was further revealed that current deflections caused by cDNA-α-HL (Figure 2, I a, b) or nDNA1_5-α-HL interactions (Figure 2, II a, b) contained two distinct levels (B1 and B2) with preserved time unfolding (B1 → B2), and the transit times corresponding to the B2 substate were shorter than those characterizing the B1 substate. In line with previous reports,29,49,50 these findings suggest that the B1 level corresponds to a single ssDNA molecule entering the α-HL’s vestibule, and the spike at the end of each B

DOI: 10.1021/acssensors.9b00553 ACS Sens. XXXX, XXX, XXX−XXX

Letter

ACS Sensors

Figure 2. Unique blockade signatures generated by ssDNAs and PNA-DNAs duplexes. Capture of single cDNA or nDNA1_5 strands inside the nanopore results in partial blockades denoted in segments in I, b (cDNA) and II, b (nDNA1_5) by B1 and B2 (see text). Such current fluctuations were further described by the typical scatter plots of the joint distributions event diagrams (event lifetime τoff vs event amplitude) in I, a (cDNA) and II, a (nDNA1_5), respectively. For clarity, the scatter diagrams were drawn from a representative experiment, for the purpose of reflecting the unique blockade signatures through an open nanopore generated by ssDNAs and PNA-DNAs duplexes. The hybridized PNA-cDNA or PNAnDNA1_5 duplexes give discernible signature events on the ionic current blockade through the nanopore, illustrated by typical traces and event diagrams (I, d and I, c - PNA-cDNA; II, d and II, c - PNA-nDNA1_5).

cDNA) = 0.87 ± 0.002 and [ΔI/IO](B2; nDNA1_5 = 0.88 ± 0.005, respectively). In the statistical evaluations above, values represent average ± SEM based upon at least 50 individual blockade readings, from at least three distinct experiments. Kinetic analysis revealed that (i) when either cDNAs or nDNA1_5s hybridized with PNA fragments in the cis chamber, the dynamics of single PNA-DNA duplexes captured inside the nanopore’s vestibule (Bc1 events) has two different pathways, with distinct event lifetimes. Such molecular steps were displayed representatively for the PNA-cDNA duplex only (Figure 2, I d; “dissociation” events and Figure 2, I d; “unzip” events, respectively); (ii) the duration of the duplex-induced Bc1 events were considerably larger than of the ssDNAs-induced B1 events. These indicate that the Bc1 substate reflects a single PNADNA (cDNA or nDNA1_5) duplex captured transiently inside the α-HL’s vestibule, before its backward passage to the cis chamber, or unzip under the electric force inside the nanopore, followed by subsequent ssDNA moiety passage across the nanopore. Through the analysis of the pooled data on the event intervals characterizing duplex dissociation without unzip (τdissociation; Figure 2, I d) and event lifetimes representing time to unzip of a vestibule-trapped PNA-DNA duplex (τunzip; Figure 2, I d) at various transmembrane potentials (ΔV), we

binding event (level B2) indicates that, after vestibule dissociation, the ssDNA passes across the constriction region of the nanopore to escape on the trans side. The single-channel current analysis revealed the emergence of additional blockade events (denoted by Bc1), recorded while stable PNA-cDNA (Figure 2, I c, d) or PNA-nDNA1_5 (Figure 2, II c, d) duplexes enter the α-HL’s vestibule. As we present in the Supporting Information, relative amplitude blockade and duration of the events shown representatively in Figure 2 were statistically analyzed, and the outcome is presented in Figure 3. From the amplitude analysis (Figure 3a,b), we noted that (i) within statistical uncertainty, the relative blockade amplitudes of the Bc1 substate were found to be similar, when either cDNA ([ΔI/IO](Bc1) = 0.417 ± 0.001) or nDNA1_5 ([ΔI/IO](Bc1) = 0.418 ± 0.005) was added in the cis chamber, and they were larger than the relative blockade amplitudes of the B1 substate induced by the ssDNA strand alone ([ΔI/IO](B1; cDNA) = 0.29 ± 0.004 and [ΔI/IO](B1; nDNA1_5) = 0.28 ± 0.007; (ii) the relative amplitude at the end of each Bc1 binding event (i.e., the Bc2 levels in Figure 2, I d and Figure 2, II d) were practically similar ([[ΔI/IO])Bc2; cDNA) = 0.85 ± 0.008 and [ΔI/IO](Bc2; nDNA1_5) = 0.89 ± 0.001) and equal to those measured in experiments with the ssDNAs strands added alone in the cis chamber (Figure 2, I b and Figure 2, II b) ([ΔI/IO](B2; C

DOI: 10.1021/acssensors.9b00553 ACS Sens. XXXX, XXX, XXX−XXX

Letter

ACS Sensors

Figure 3. Blockade amplitude and kinetic description of blockade events induced by ssDNAs or PNA-DNA duplexes. Relative current blockades characterizing the subconductance levels associated with the α-HL-cDNA and α-HL-PNA-cDNA interactions (a), or α-HL-nDNA1_5 and α-HLPNA-nDNA1_5 interactions (b), as a function of the applied voltage. The lines represent linear fits with zero slope. A single nucleotide mismatch in the PNA-nDNA1_5 duplex (d), as compared to the fully complementary strands in the PNA-cDNA complex (c), induces an approximately 1 order of magnitude shortening of the averaged event lifetimes (τ̅off) of corresponding duplexes inside the nanopore’s vestibule, at all applied ΔVs. 1 The average τoff ̅ = k unzip + kdissociation , and the rate constants kunzip and kdissociation reflect the duplex unzip and dissociation from nanopore without unzip, respectively. Data statistics was performed on blockade events from at least three independent experiments.

derived corresponding averaged values (τ̅off) (Figure 3c,d). As proposed previously,45 the average τ̅off is related to the rate constants describing with the duplex unzip (kunzip) and duplex dissociation from nanopore without unzip (kdissociation) 1 (τoff ). ̅ = k +k unzip

In summary, we proved that electrical recordings through the wild-type α-HL nanopore can be used for label-free, realtime detection of short ssDNAs by exploiting the hybridization reaction with complementary PNAs. We observed two different pathways of the PNA-DNA duplex dissociation from the nanopore’s vestibule, reflecting unbinding events proceeding with and without duplex unzip. As the positions of mismatches along the duplex alters their sensitivity of detection,51 and the mismatch has the most dramatic effect when it was positioned in the middle of the oligonucleotide,38 the demonstration provided herein regarding the detection of mismatches engineered at one end of the ssDNAwhere a lesser sensitivity of detection is expected substantiate the usefulness of the presented approach to study DNA duplex formation and pinpoint mismatches. To optimize the presented undertaking, it may be argued in favor of employing shorter PNAs and complementary ssDNAs sequences; however, this may present technical challenges, as duplexes of 7 or 8 base pairs already dissociate within a second.52 As a prospective use of this approach in point-of-care units, integrating nanomanufacturing technologies enabling solidstate or protein-based nanopores coupled tightly with microelectronics CMOS amplifiers and controlled by cloud-based software applications have the potential of setting up viable solutions for portable miniaturized devices enabling fast genotyping or screening exogenous DNAs.

dissociation

As cDNA and nDNA1_5 have similar lengths, the overall electric charge of the corresponding PNA-DNA duplexes is similar. Thus, for both PNA-cDNA and PNA-nDNA1_5 duplexes, the reaction constants describing dissociation from nanopore without unzip (kdissociation) are similar and energetically unfavored by the ΔV, as they reflect the passage of similarly charged complexes backwardly to the cis side (insets of Figure 3c,d). The rate constant describing the duplex unzip (kunzip) becomes larger at increasing ΔV values, as larger electric forces acting on the duplex favor its zippering rate.30,31 In addition, the presence of the mismatch between the two strands in the duplex results in a correspondingly larger kunzip (insets of Figure 3c,d).48 Applied to our case, as kunzip (PNA-cDNA) < kunzip (PNA-nDNA1_5) and kdissociation (PNA-cDNA) ≈ kdissociation (PNA-nDNA1_5) at any ΔV, τ̅off (PNA-cDNA) should be greater than τ̅off (PNA-nDNA1_5). This is accurately reflected by the experimental data (Figure 3c,d). The simple assay undertaken herein demonstrates that the free energy change brought about by a nucleotide mismatch in the PNA-DNA duplex is readily seen from the statistical analysis of the pooled dissociation times of the duplex from the α-HL’s vestibule, as an order of magnitude increase in the τ̅off (PNAcDNA) vs τ̅off (PNA-nDNA1_5). D

DOI: 10.1021/acssensors.9b00553 ACS Sens. XXXX, XXX, XXX−XXX

Letter

ACS Sensors

■

Acid, Polyadenylic Acid, and Polyuridylic Acid as Homopolymers or as Segments within Single RNA Molecules. Biophys. J. 1999, 77, 3227−3233. (9) Cao, C.; Ying, Y. L.; Hu, Z. L.; Liao, D. F.; Tian, H.; Long, Y. T. Discrimination of Oligonucleotides of Different Lengths with a WildType Aerolysin Nanopore. Nat. Nanotechnol. 2016, 11, 713−718. (10) Wang, Y. Q.; Cao, C.; Ying, Y. L.; Li, S.; Wang, M. B.; Huang, J.; Long, Y. T. Rationally Designed Sensing Selectivity and Sensitivity of an Aerolysin Nanopore via Site-Directed Mutagenesis. ACS Sens. 2018, 3, 779−783. (11) Zwolak, M.; Di Ventra, M. Colloquium: Physical Approaches to DNA Sequencing and Detection. Rev. Mod. Phys. 2008, 80, 141−165. (12) Howorka, S.; Siwy, Z. Nanopore Analytics: Sensing of Single Molecules. Chem. Soc. Rev. 2009, 38, 2360−2384. (13) Strezoska, Z.; Paunesku, T.; Radosavljevic, D.; Labat, I.; Drmanac, R.; Crkvenjako, R. DNA Sequencing by Hybridization: 100 Bases Read by a Non-Gel-Based Method. Proc. Natl. Acad. Sci. U. S. A. 1991, 88, 10089−10093. (14) Liu, F.; Jenssen, T.-K.; Trimarchi, J.; Punzo, C.; Cepko, C. L.; Ohno-Machado, L.; Hovig, E.; Kuo, W. P. Comparison of Hybridization-based and Sequencing-based Gene Expression Technologies on Biological Replicates. BMC Genomics 2007, 8, 153. (15) Gasc, C.; Peyretaillade, E.; Peyret, P. Sequence Capture by Hybridization to Explore Modern and Ancient Genomic Diversity in Model and Nonmodel Organisms. Nucleic Acids Res. 2016, 44, 4504− 4518. (16) Okou, D. T.; Steinberg, K. M.; Middle, C.; Cutler, D. J.; Albert, T. J.; Zwick, M. E. Microarray-Based Genomic Selection for Highthroughput Resequencing. Nat. Methods 2007, 4, 907−909. (17) Shigemizu, D.; Momozawa, Y.; Abe, T.; Morizono, T.; Boroevich, K. A.; Takata, S.; Ashikawa, K.; Kubo, M.; Tsunoda, T. Performance Comparison of Four Commercial Human Whole-Exome Capture Platforms. Sci. Rep. 2015, 5, 12742. (18) Sosnowski, R.; Heller, M. J.; Tu, E.; Forster, A. H.; Radtkey, R. Active Microelectronic Array System for DNA Hybridization, Genotyping and Pharmacogenomic Applications. Psychiatr. Genet. 2002, 12, 181−192. (19) McCord, B. R.; Gauthier, Q.; Cho, S.; Roig, M. N.; GibsonDaw, G. C.; Young, B.; Taglia, F.; Zapico, S. C.; Fogliota Mariot, R.; Lee, S. B.; Duncan, G. Forensic DNA Analysis. Anal. Chem. 2019, 91, 673−688. (20) Khier, S.; Lohan, L. Kinetics of Circulating Cell-Free DNA for Biomedical Applications: Critical Appraisal of the Literature. Future Sci. OA 2018, 4, No. FSO295. (21) Hall, J. M.; Lee, M. K.; Newman, B.; Morrow, J. E.; Anderson, L. A.; Huey, B.; King, M. C. Linkage of Early-onset Familial Breast Cancer to Chromosome 17q21. Science (Washington, DC, U. S.) 1990, 250, 1684−1689. (22) Thierry, A. R.; Mouliere, F.; El Messaoudi, S.; Mollevi, C.; Lopez-Crapez, E.; Rolet, F.; Gillet, B.; Gongora, C.; Dechelotte, P.; Robert, B.; Del Rio, M.; Lamy, P. J.; Bibeau, F.; Nouaille, M.; Loriot, V.; Jarrousse, A. S.; Molina, F.; Mathonnet, M.; Pezet, D.; Ychou, M. Clinical Validation of the Detection of KRAS and BRAF Mutations from Circulating Tumor DNA. Nat. Med. 2014, 20, 430−435. (23) Rizzi, G.; Lee, J.-R.; Dahl, C.; Guldberg, P.; Dufva, M.; Wang, S. X.; Hansen, M. F. Simultaneous Profiling of DNA Mutation and Methylation by Melting Analysis Using Magnetoresistive Biosensor Array. ACS Nano 2017, 11, 8864−8870. (24) Erali, M.; Voelkerding, K. V.; Wittwer, C. T. High Resolution Melting Applications for Clinical Laboratory Medicine. Exp. Mol. Pathol. 2008, 85, 50−58. (25) Jung, Y. K.; Kim, J.; Mathies, R. A. Microfluidic Hydrogel Arrays for Direct Genotyping of Clinical Samples. Biosens. Bioelectron. 2016, 79, 371−378. (26) Pinzani, P.; Salvianti, F.; Cascella, R.; Massi, D.; De Giorgi, V.; Pazzagli, M.; Orlando, C. Allele Specific Taqman-Based Real-Time PCR Assay to Quantify Circulating BRAFV600E Mutated DNA in Plasma of Melanoma Patients. Clin. Chim. Acta 2010, 411, 1319− 1324.

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssensors.9b00553. Reagents and chemicals, as well as experimental methods used (PDF)

■

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Tudor Luchian: 0000-0002-9388-7266 Author Contributions

# A.A. and L.M. contributed equally. T.L. and Y.P. conceived the idea and designed the experiments; L.M. and A.A. performed the nanopore experiments; L.M., A.A., and J.P. analyzed the experimental data and interpreted the data; T.L. and L.M. cowrote the paper; Y.P, T.L., and C.H.S. supervised the project. All authors have given approval to the final version of the manuscript.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The work was supported by National Research Foundation of Korea (NRF) (2016R1A2A1A05005440), Global Research Laboratory (GRL) (NRF-2014K1A1A2064460), IITP grant (MSIT) (2017-0-01714), and grants PN-III-P4-ID-PCE-20160026 and PN-IIIP1-1.1-TE-2016-0508.

■

REFERENCES

(1) Kasianowicz, J. J.; Brandin, E.; Branton, D.; Deamer, D. W. Characterization of Individual Polynucleotide Molecules Using a Membrane Channel. Proc. Natl. Acad. Sci. U. S. A. 1996, 93, 13770− 13773. (2) Branton, D.; Deamer, D. W.; Marziali, A.; Bayley, H.; Benner, S. A.; Butler, T.; Di Ventra, M.; Garaj, S.; Hibbs, A.; Huang, X.; Jovanovich, S. B.; Krstic, P. S.; Lindsay, S.; Ling, X. S.; Mastrangelo, C. H.; Meller, A.; Oliver, J. S.; Pershin, Y. V.; Ramsey, J. M.; Riehn, R.; et al. The Potential and Challenges of Nanopore Sequencing. Nat. Biotechnol. 2008, 26, 1146−1153. (3) Stoddart, D.; Heron, A.; Mikhailova, E.; Maglia, G.; Bayley, H. Single Nucleotide Discrimination in Immobilized DNA Oligonucleotides with a Biological Nanopore. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 7702−7707. (4) Cao, C.; Yu, J.; Li, M. Y.; Wang, Y. Q.; Tian, H.; Long, Y.-T. Direct Readout of Single Nucleobase Variations in an Oligonucleotide. Small 2017, 13, 1702011. (5) Mitchell, N.; Howorka, S. Chemical Tags Facilitate the Sensing of Individual DNA Strands with Nanopores. Angew. Chem., Int. Ed. 2008, 47, 5565−5568. (6) Manrao, E. A.; Derrington, I. M.; Laszlo, A. H.; Langford, K. W.; Hopper, M. K.; Gillgren, N.; Pavlenok, M.; Niederweis, M.; Gundlach, J. H. Reading DNA at Single-Nucleotide Resolution with a Mutant MspA Nanopore and phi29 DNA Polymerase. Nat. Biotechnol. 2012, 30, 349−353. (7) An, N.; Fleming, A. M.; White, H. S.; Burrows, C. J. Crown Ether−Electrolyte Interactions Permit Nanopore Detection of Individual DNA Abasic Sites in Single Molecules. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 11504−11509. (8) Akeson, M.; Branton, D.; Kasianowicz, J. J.; Brandin, E.; Deamer, D. W. Microsecond Time-Scale Discrimination among Polycytidylic E

DOI: 10.1021/acssensors.9b00553 ACS Sens. XXXX, XXX, XXX−XXX

Letter

ACS Sensors (27) Drmanac, S.; Kita, D.; Labat, I.; Hauser, B.; Schmidt, C.; Burczak, J. D.; Drmanac, R. Accurate Sequencing by Hybridization for DNA Diagnostics and Individual Genomics. Nat. Biotechnol. 1998, 16, 54−58. (28) Abi, A.; Safavi, A. Targeted Detection of Single-Nucleotide Variations: Progress and Promise. ACS Sens. 2019, 4, 792. (29) Howorka, S.; Movileanu, L.; Braha, O.; Bayley, H. Kinetics of Duplex Formation for Individual DNA Strands within a Single Protein Nanopore. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 12996−13001. (30) Mathé, J.; Visram, H.; Viasnoff, V.; Rabin, Y.; Meller, A. Nanopore Unzipping of Individual DNA Hairpin Molecules. Biophys. J. 2004, 87, 3205−3212. (31) Wang, Y.; Tian, K.; Hunter, L. L.; Ritzo, B.; Gu, L.-Q. Probing Molecular Pathways for DNA Orientational Trapping, Unzipping and Translocation in Nanopores by Using a Tunable Overhang Sensor. Nanoscale 2014, 6, 11372−11379. (32) Balagurusamy, V. S. K.; Weinger, P.; Ling, X. S. Detection of DNA Hybridizations Using Solid-State Nanopores. Nanotechnology 2010, 21, 335102. (33) Perera, R. T.; Fleming, A. M.; Peterson, A. M.; Heemstra, J. M.; Burrows, C. J.; White, H. S. Unzipping of A-Form DNA-RNA, AForm DNA-PNA, and B-Form DNA-DNA in the α-Hemolysin Nanopore. Biophys. J. 2016, 110, 306−314. (34) Lin, Y.; Shi, X.; Liu, S.-C.; Ying, Y.-L.; Li, Q.; Gao, R.; Fathi, F.; Long, Y.-T.; Tian, H. Characterization of the DNA Duplex Unzipping through a sub-2 nm Solid-State Nanopore. Chem. Commun. 2017, 53, 3539−3542. (35) Ali, M.; Neumann, R.; Ensinger, W. Sequence Specific Recognition of DNA Oligomer Using Peptide Nucleic Acid (PNA)Modified Synthetic Ion Channels: PNA/DNA Hybridization in Nanoconfined Environment. ACS Nano 2010, 4, 7267−7274. (36) Morin, T. J.; Shropshire, T.; Liu, X.; Briggs, K.; Huynh, C.; Tabard-Cossa, V.; Wang, H.; Dunbar, W. B. Nanopore-Based Target Sequence Detection. PLoS One 2016, 11, No. e0154426. (37) Loh, A. Y. Y.; Burgess, C. H.; Tanase, D. A.; Ferrari, G.; McLachlan, M. A.; Cass, A. E. G.; Albrecht, T. Electric SingleMolecule Hybridization Detector for Short DNA Fragments. Anal. Chem. 2018, 90, 14063−14071. (38) Howorka, S.; Cheley, S.; Bayley, H. Sequence-Specific Detection of Individual DNA Strands Using Engineered Nanopores. Nat. Biotechnol. 2001, 19, 636−639. (39) Tian, K.; He, Z.; Wang, Y.; Chen, S.-J.; Gu, L.-Q. Designing a Polycationic Probe for Simultaneous Enrichment and Detection of MicroRNAs in a Nanopore. ACS Nano 2013, 7, 3962−3969. (40) Wu, X.; Guo, B.; Sheng, Y.; Zhang, Y.; Wang, J.; Peng, S.; Liu, L.; Wu, H.-C. Multiplexed Discrimination of microRNA Single Nucleotide Variants through Triplex Molecular Beacon Sensors. Chem. Commun. 2018, 54, 7673−7676. (41) Wang, Y.; Zheng, D.; Tan, Q.; Wang, M. X.; Gu, L.-Q. Nanopore-Based Detection of Circulating microRNAs in Lung Cancer Patients. Nat. Nanotechnol. 2011, 6, 668−674. (42) Wanunu, M.; Dadosh, T.; Ray, V.; Jin, J. M.; McReynolds, L.; Drndic, M. Rapid Electronic Detection of Probe-Specific microRNAs Using Thin Nanopore Sensors. Nat. Nanotechnol. 2010, 5, 807−814. (43) Wang, L.; Chen, X.; Zhou, S.; Roozbahani, G. M.; Zhang, Y.; Wang, D.; Guan, X. Displacement Chemistry-Based Nanopore Analysis of Nucleic Acids in Complicated Matrices. Chem. Commun. 2018, 54, 13977−13980. (44) Gu, L.-Q.; Wanunu, M.; Wang, M. X.; McReynolds, L.; Wang, Y. Detection of miRNAs with a Nanopore Single-Molecule Counter. Expert Rev. Mol. Diagn. 2012, 12, 573−584. (45) Ciuca, A.; Asandei, A.; Schiopu, I.; Apetrei, A.; Mereuta, L.; Seo, C.-H.; Park, Y.; Luchian, T. Single Molecule, Real-Time Dissecting of Peptide Nucleic Acids-DNA Duplexes with a Protein Nanopore Tweezer. Anal. Chem. 2018, 90, 7682−7690. (46) Egholm, M.; Buchardt, O.; Christensen, L.; Behrens, C.; Freier, S. M.; Driver, D. A.; Berg, R. H.; Kim, S. K.; Norden, B.; Nielsen, P. E. PNA Hybridizes to Complementary Oligonucleotides Obeying the Watson-Crick Hydrogen-Bonding Rules. Nature 1993, 365, 566−568.

(47) Tomac, S.; Sarkar, M.; Ratilainen, T.; Wittung, P.; Nielsen, P. E.; Nordén, B.; Gräslund, A. Ionic Effects on the Stability and Conformation of Peptide Nucleic Acid Complexes. J. Am. Chem. Soc. 1996, 118, 5544−5552. (48) Cisse, I. I.; Kim, H.; Ha, T. A Rule of Seven in Watson-Crick Base-Pairing of Mismatched Sequences. Nat. Struct. Mol. Biol. 2012, 19, 623−627. (49) Mereuta, L.; Roy, M.; Asandei, A.; Lee, J. K.; Park, Y.; Andricioaei, I.; Luchian, T. Slowing Down Single-Molecule Trafficking through a Protein Nanopore Reveals Intermediates for Peptide Translocation. Sci. Rep. 2015, 4, 3885. (50) Vercoutere, W.; Winters-Hilt, S.; Olsen, H.; Deamer, D.; Haussler, D.; Akeson, M. Rapid Discrimination among Individual DNA Hairpin Molecules at Single-Nucleotide Resolution Using an Ion Channel. Nat. Biotechnol. 2001, 19, 248−252. (51) McNally, B.; Wanunu, M.; Meller, A. Electro-Mechanical Unzipping of Individual DNA Molecules Using Synthetic sub-2 nm Pores. Nano Lett. 2008, 8, 3418−3422. (52) Mosayebi, M.; Louis, A. A.; Doye, J. P. K.; Ouldridge, T. E. Force-Induced Rupture of a DNA Duplex: From Fundamentals to Force Sensors. ACS Nano 2015, 9, 11993−12003.

F

DOI: 10.1021/acssensors.9b00553 ACS Sens. XXXX, XXX, XXX−XXX