Nanopore-Assisted, Sequence-Specific Detection and Single

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Nanopore-Assisted, Sequence-Specific Detection and SingleMolecule Hybridization Analysis of Short, Single-Stranded DNAs Loredana Mereuta, Alina Asandei, Irina Schiopu, Yoonkyung Park, and Tudor Luchian Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b02080 • Publication Date (Web): 04 Jun 2019 Downloaded from http://pubs.acs.org on June 5, 2019

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Analytical Chemistry

Nanopore-Assisted, Sequence-Specific Detection and Single-Molecule Hybridization Analysis of Short, Single-Stranded DNAs Loredana Mereuta1,#, Alina Asandei2,#, Irina Schiopu2, Yoonkyung Park3,*, Tudor Luchian1,*. 1 Department

of Physics, ‘Alexandru I. Cuza’ University, Iasi, Romania, 700506 Research Institute, Sciences Department, ‘Alexandru I. Cuza’ University, Iasi, Romania, 700506 3 Department of Department of Biomedical Science and Research Center for Proteinaceous Materials (RCPM), Chosun University, Gwangju, Republic of Korea, 61452. 2 Interdisciplinary

ABSTRACT: We report here on the ability of the α-hemolysin (α-HL) nanopore to achieve label-free, selective and real-time detection of 15 nt long ssDNA fragments detection in solution, by exploiting their hybridization with freely added, polycationic peptides-functionalized PNAs. At the core of our work lies the paradigm that when PNAs and ssDNA are mixed together, the bulk concentration of free PNA decreases, depending upon the (mis)match degree between complementary strands and their relative concentrations. We demonstrate that ssDNA sensing principle and throughput of the method are determined by the rate at which nonhybridized, polycationic peptides-functionalized PNA molecules, arrive at the α-HL’s vestibule entrance and thread into the nanopore. We found that with the application of a 30-fold salt gradient across the nanopore, the method enhances single-molecule detection sensitivity in the nanomolar range of ssDNA concentrations. This study demonstrates that the transmembrane potential-dependent unzip of single PNA-DNA duplexes at the α-HL’s β-barrel entry, permits the discrimination between sequences that differ by one base pair.

With the presentation of conductance studies through individual nanopores more than two decades ago, it came the prospect for a cheaper, versatile, easier-to-implement and run method, to revealing properties of matter at the single-molecule level.1-10 The cornerstone of the approach was represented by pioneering studies by Kasianowicz and Bezrukov,11,12 revealing that αhemolysin (α-HL) protein nanopore of Staphylococcus aureus permits the investigation of reactions dynamic. Soon after, the idea of using nanopores to sequence DNA arose.13-27 Despite considerable advances, major challenges plaguing the accurate sequencing of DNA with nanopores lie in: (i) the spatial resolution provided by the nanopore, and (ii) translocation velocity (1–3 µs/nt)28 determined mainly by electrophoresis, hydrodynamic friction, and surface adsorption effects 29-31 which, unimpeded, imposes severe constrains in detecting single nucleotides passage across the nanopore. There are, however, many applications which do not require sequencing per se, but instead need to detect a specific DNA target.32-38 For such tasks, hybridization assays, including microarrays39 or qPCR40 are usually employed for achieve sequence detection. These methods do present limitations, however, caused by nonspecific amplification or hybridization, insufficient base selectivity and sensitivity, costly infrastructure and complex work.39-43 Alternatively, nanomaterials were employed for fluorescence assays of nucleic acids.44,45 Other approaches for detecting hybridization of short DNAs based particularly on nanoparticles, were proven useful for multiplexed detection of nucleic acids,46,47 while others have demonstrated the ability of polyelectrolyte-modified capacitive EIS sensors for the detection of DNA hybridization in aqueous buffers.48 Single-

molecule, fluorescence resonance energy transfer (smFRET) and fluorescence correlation spectroscopy methods, were also used to detect duplex formation within or between individual DNA strands.43,49 Nanopores presented an attractive approach to probing DNA strands by selective hybridization with DNA or PNA targets.5061 Due to the fact that short nucleic acids fragments (9-12 bases) bind transiently with their complementary strands at room temperature,62 electrical force applied on the nucleic acid dimer inside the nanopore, easily unzips the molecule. This imparts the nanopore approach with the ability to also quantify in realtime the DNA unzipping kinetics, from single-molecule kinetics analysis the dwell-times of the molecule inside the nanopore,50,58,63-65 which is crucial for nanopore-based single nucleotide polymorphism detection. This approach was highly successful for the task of miRNA detection66 or selective detection of miRNAs with single nucleotide variants.67-70 Here, we combine the use of wild type α-HL homo-oligomer nanopores with polycationic peptide-functionalized PNA monomers, allowing duplex formation with complementary sequences, to detect short ssDNA fragments at the singlemolecule level. Statistical quantification of the DNA-PNA hybridization and of DNA-PNA duplex rupture at the singlemolecule level, led to measurable properties including detection propensity of specific ssDNA sequences by the target PNA, hybridization energy, and base pair mismatches. Efficiencies of mismatch discrimination using size-varied captured ssDNA probes were examined.

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Experimental Section Materials. The polypeptide-functionalized (PNA) and ssDNA sequences were synthesized and purified by Panagene Inc., Republic of Korea (PNA) and Sigma-Aldrich, Germany (ssDNAs), according to the sequences designed by us. The primary sequences and molecular weights of the PNA and ssDNAs used herein are shown in Table 1. Table 1 Primary sequences of the polypeptidefunctionalized PNA and ssDNA strands. The red-marked region within the nDNA1_5, nDNA3_5, nDNA3_3, nDNA7_5 represents the mismatches introduced to the complementary domain (cDNA) relative to the PNA sequence. All sequences were aligned as to readily visualize the base-pairs from the opposite strands. Polynucleotid e PNA

Primary sequence Ac-(R)10-5’- GTGATATACGGTGAT-3’

Mw (g/mol) 5760.9

cDNA

3’-CACTATATGCCACTA-5’

4499

nDNA1_5

3’-CACTATATGCCACTT-5’

4490

nDNA3_5

3’-CACTATATGCCATAT-5’

4514

nDNA3_3

3’-TTATATATGCCACTA-5’

4529

nDNA7_5

3’-CACTATATTTGTGAT-5’

4560

The buffer solutions and sample preparation are detailed in the Supporting Information. Single-molecule electrophysiology. The electrophysiology experiments were performed in a recording chamber separated in two compartments (denoted by cis - grounded and trans) by a 25 µm-thick Teflon film (Goodfellow, Malvern, MA, USA), that contained a punctured aperture of about 120 µm in diameter, for the planar lipid membrane formation. Using the Montal-Muller technique65,71 the bilayers were formed across the aperture from the dissolved lipids in pentane, which were added on the surface of the electrolyte solution in both compartments. About 0.5 – 2 μL of the α-HL was added to the grounded, cis-compartment, from a stock solution made in 0.5 M KCl. After the insertion of a single heptameric α-HL nanopore into the membrane, and depending on the particular experiment, ssDNAs and PNA were added from the stock solution to either the cis- or trans-side of the membrane. Because of the slight anion selectivity of the α-HL at neutral pH (PK+/PCl- ~ 0.7), when the electrolyte concentration gradient was applied across the membrane, a small reversal potential was observed (Vreversal = 8.2 mV).72 To mitigate the effect of the reversal potential upon the net transmembrane voltage sensed by the nanopore, prior to applying the transmembrane voltage, the reversal potential was nullified from the amplifier’s voltage offset knob. The ionic currents were recorded with an Axopatch 200B (Molecular Devices, USA) or EPC-8 (HEKA, Germany) amplifier, low-pass filtered at 10 kHz and digitized with a NI PCI 6221, 16-bit acquisition board (National Instruments, USA) at a sampling frequency of 50 kHz. The statistical analysis on the relative blockage amplitudes on the electric current through a single α-HL protein, as well as the frequency and duration of the PNA- or DNA-PNA-induced current blockades were analyzed within the statistics of exponentially distributed events, as previously described.73 To quantify the blockade probabilities of PNA-nanopore interactions in the absence or presence of distinct ssDNAs, all-points histograms were constructed based on the ionic current recordings, leading

to values of block probability = 𝐴𝑏𝑙𝑜𝑐𝑘 + 𝐴𝑜𝑝𝑒𝑛 (𝐴𝑏𝑙𝑜𝑐𝑘 and 𝐴𝑜𝑝𝑒𝑛 refer to the histogram areas assigned to the ‘block’ and ‘open’ states, respectively, on the corresponding histogram). Graphic representations and numerical analysis of the recorded data were done in Origin 6 (OriginLab, Northampton, MA, USA) and pClamp 6.03 (Molecular Devices, USA) software. Results and Discussion Principle of the experiment Unlike previous studies,21,50,55,63 a conceptually different approach was taken here to detecting ssDNAs with the α-HL nanopore. Instead of directly capturing ssDNAs, we monitored the decrease of the aqueous concentration of a complementary cationic polypeptide-functionalized PNA sequence, following selective DNA-PNA hybridization (Fig. 1). With potential applicability to discoveries presented herein, in early work authors have shown that by engineering a specific analyte recognition site on selected polymers, the nanopore-based single-molecule detection method has the potential of simultaneous detection and quantitation of multiple analytes.74 As the electrophoretic force needed to capture the functionalized PNA molecules inside the α-HL vestibule, drives the anionic ssDNA fragments away from the nanopore,65,66 the approach seems ideally suited in applications seeking selective, individual ssDNA species detection within a mixture of other ssDNAs or anionic molecules, with a high signal-to-noise ratio. As reported initially,75 to optimize such capture events leading to indirect ssDNA detection, we added all molecules on the cis side of the membrane. The slightly larger vestibule opening of the α-HL on the cis side (diameter of ~ 2.6 nm) than its β-barrel on the trans side (diameter of ~ 2 nm)75,76 provides a reduced entropy barrier for molecules partitioning within the α-HL. A similar strategy was reported in subsequent work.77 Electrical detection of ssDNAs hybridization by polypeptide-functionalized PNAs In Fig. 1 we illustrate that cis-side added, cationic polypeptidefunctionalized PNAs get captured by a single α-HL clamped at trans-negative (b), seen as reversible current blockades through the nanopore. The voltage-dependent kinetic behavior of PNAα-HL interactions is described in Fig. S1. Trans-positive potentials on the other hand, preclude PNA-α-HL nanopore interactions, indicated by the absence of reversible blockade events (Fig. 1, g)

Fig.1 Nanopore-based detection of bulk PNAs depletion through hybridization with ssDNA fragments, allows accurate and real-time detection of probe ssDNA. (a) At ΔV = -180 mV, anionic ssDNA fragments (cDNA) added on the cis


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side (0.05 µM) of the lipid membrane containing a single α-HL, are driven away from the nanopore. (b) Cis-added polycationic peptide-functionalized PNAs (5 µM) are electrophoretically captured at the α-HL’s vestibule at ΔV = -180 mV, leading to reversible conductance changes of the nanopore (upwardly oriented events) (block probability PNA = 0.66). (c) When the cis-added PNAs are mixed with perfectly matched ssDNAs (cDNA) (PNA:cDNA; molar ratio of 1:0.05), a reduction in the PNA-induced blockade events ensues. Addition of ssDNAs containing three (d, nDNA3_5) and respectively seven mismatched bases toward the PNA (e, nDNA7_5), leave the frequency of PNA-induced blockade events only slightly modified. At ΔV = + 180 mV, cis-added cDNAs are reversibly captured at the α-HL’s vestibule entry (downwardly oriented spikes, f), whereas polycationic peptide-functionalized PNAs are precluded from capture (g). Further, at ΔV = + 180 mV, cisside mixing of PNA and cDNA at a molar ratio of 1:0.05 (h), results in distinct blockade events reflecting the nanopore capture of anionic PNA-cDNA duplexes (level ‘#’) or singlestranded cDNAs (level ‘*’). In contrast, PNAs mixing with the three- (i, nDNA3_5) or seven-mismatched ssDNAs (j, nDNA7_5) resulted in blockade events suggestive of sole ssDNAs interaction with the nanopore (level ‘*’).

The hybridization-driven, duplex formation between cis-added ssDNAs and target PNAs, leads to the depletion of bulk PNA concentration. Given the net negative charge on the DNA-PNA duplex (bare charge of - 5│e-│) and ssDNA fragments (bare charge of -15│e-│), at trans-negative potentials only the free, non-hybridized cationic polypeptide-functionalized PNAs will be captured electrophoretically by the nanopore (Fig. 1, b-e). As for a second-order reaction, the PNA-nanopore association rate depends on the PNA’s bulk concentration, which in our experiments lowers due hybridization. We anticipated that subsequent to ssDNA-PNA hybridization in the cis chamber, a less frequent appearance of free PNA-induced blockade events will ensue, as compared to that measured before hybridization. In accordance to this, we found that addition of the complementary cDNA (0.25 µM) on the cis chamber containing PNA (5 µM), led to an almost cessation in the occurrence of blockade events at ΔV = -180 mV (Fig. 1, c; block probability PNA:cDNA = 0.003), as compared to the case when cDNA was absent (Fig. 1, b; block probability PNA = 0.66). Furthermore, as shown in Fig. 1, d and Fig. 1, e, addition of nDNA3_5 or nDNA7_5, respectively, barely affects the likelihood of PNAinduced blockade events on the nanopore as compared to the control case, and the phenomenon is correlated with the degree of base mismatch between the ssDNAs and PNA strands (PNA alone; Fig. 1, b) (block probability PNA:nDNA3_5 = 0.62, block probability PNA:nDNA7_5 = 0.51). We noted that following PNA-cDNA hybridization, initially mixed at a molar ratio of 1:0.05, very few to none PNA-induced blockade events ensued at ΔV = -180 mV (Fig. 1, c). This is unexpected, as non-hybridized PNAs would still be available for capture at the α-HL’s vestibule, as evidenced during experiments involving the nDNA3_5 and nDNA7_5 constructs (Fig. 1, d, e). To investigate this further, we assessed the analyte-nanopore interactions at positive potentials of ΔV = +180 mV, when the cis chamber contained solely cDNA (Fig. 1, f), or PNA:cDNA (Fig. 1, h), PNA:nDNA3_5 (Fig. 1, i) or

PNA:nDNA7_5 (Fig. 1, j) mixed initially at a molar ratio of 1:0.05. The blockade activity recorded under the above conditions (Fig. 1, f, h-j), demonstrates that apparent cessation of binding events to the nanopore at ΔV = -180 mV following cDNA-PNA hybridization (Fig. 1, c), does not constitute an experimental artifact or irreversible collapse of the nanopore. At the present moment we lack a clear explanation for the phenomenon embodied by Fig. 1, c, and aim for an in-depth follow-up of this investigation in upcoming work. The presented model of PNA-nanopore interactions following hybridization with various ssDNAs, was further tested. As shown in Fig. 2, incremental, cis-side addition of various concentrations from the cDNA (I, a-c), nDNA3_5 (II, a-c) and nDNA7_5, respectively (III, a, b), led to a corresponding decrease in the blockade probability of events reflecting PNAnanopore interactions at ΔV = -180 mV, and the prevalence of the effect is sensitive to the degree of DNA-PNA base mismatch.

Fig. 2 Concentration-dependent effect of distinct ssDNAs on the blockade events reflecting PNA-nanopore interactions. Typical traces recorded at ΔV = -180 mV and corresponding all-points histograms, showing the effect of distinct relative concentrations of cDNA (I, a-c), nDNA3_5 (II, a-c) and nDNA7_5, respectively (III, a, b), relative to PNA (5 µM), on PNA-induced ion current blockades through a single α-HL nanopore. All nucleic acid sequences were added on the cis side of the membrane. With the addition of nDNA7_5 fragments (III), a visible effect of PNA depletion was absent even at the largest molar ratio tested (PNA:nDNA7_5) / (1:0.05). The molecular events related to PNA-nanopore interactions before ssDNA-induced hybridization (Fig. 2 (I-III)), were quantified through the block probability PNA = 0.57. Subsequent cDNA addition at distinct molar ratios relative to PNA’s concentration, led to values of block probability PNA:cDNA/1:0.01 = 0.46 (Fig. 2, I, b) and block probability PNA:cDNA/1:0.05 = 0.004 (Fig. 2, I, c), and this phenomenon is dependent on the degree of PNA:ssDNA base mismatch (block probability PNA:nDNA3_5/1:0.01 = 0.38, Fig. 2, II, b; block probability PNA:nDNA3_5/1:0.05 = 0.18; Fig. 2, II, c; block probability PNA:nDNA7_5/ 1:0.05 = 0.47; Fig. 2, III, b). Thus far, the findings showcase a relevant strength of the approach, namely to achieve fast detection of a known ssDNA


Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

sequence from other fragments in aqueous solution, via progressive PNA depletion following DNA-PNA hybridization, without the need of additional separation or purification steps. DNA-PNA hybridization was found sensitive to the degree of base complementarity and on the position of the mismatch along the duplex, which is in good agreement with previous reports.58 We illustrate this in Fig. S2, showing that three bases mismatches engineered at the opposite ends of the ssDNA fragment (compare nDNA3_3 vs. nDNA3_5), visibly influence the ssDNA propensity to deplete bulk PNA concentration via hybridization. A salt gradient enhances the nanopore sensitivity of the ssDNA detection To develop this approach into a viable ssDNA fingerprinting technology, for future real-world analytical applications, the detection threshold should match the low concentration of ssDNAs in biological fluids; for instance, the concentration of free circulating DNA in plasma and serum is in the range of 1.8 ÷ 35 ng ml-1.78 In the present approach, the (indirect) ssDNA sensing ability is limited by the lowest concentration of PNA which can be optimally detected with the nanopore, and this creates a challenge. If the objective is to increase the detection limit of the probe ssDNA, from the simple bimolecular model of ssDNA-PNA hybridization it is optimal to start with a correspondingly low concentration of the PNA. Namely, in a ssDNA- PNA mixture, once the ssDNA-PNA hybridization achieves equilibrium resulting in duplexes with the concentration [𝑃𝑁𝐴:𝐷𝑁𝐴]𝑒𝑞., the value of [𝑃𝑁𝐴:𝐷𝑁𝐴]𝑒𝑞. relative to the initial concentration of PNA [PNA0], expressed

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nanopore’ in the Supporting Information), to augment the electrophoretic capture of the analyte by the nanopore.79,80 The representative experiment illustrated in Fig.3, I, demonstrates that a salt gradient across the nanopore (3 M KCl (trans):0.1 M KCl (cis)), leads to an optimal capture rate of cisside added polycationic peptides-functionalized PNAs (0.5 µM), and the process was visible at lower transmembrane potentials (ΔV = -160 mV) as compared to previously shown data. In a quantitative view, the average number of PNAinduced blockade events grew from 3 events/minute (Fig. 3, I, a,b; symmetrical salt, [PNA] = 3 µM ) to about 10 events/minute (Fig. 3, I, c,d; asymmetrical salt, [PNA] = 0.5 µM). Further addition on the cis side of the perfectly matched cDNA sequence at increasing concentrations of 0.005 µM, 0.01 µM and 0.03 µM, resulted in a corresponding increase of time intervals measured between successive PNA-induced blockade events (τon) (Fig. 3, II).

[𝑃𝑁𝐴:𝐷𝑁𝐴]𝑒𝑞.

as percentage values ( [𝑃𝑁𝐴0] x 100), is augmented when PNA0 has correspondingly lower values (see ‘Quantitative description of the DNA-PNA hybridization, within the Langmuir binding model with depletion’ in the Supporting Information). In other words, under conditions of low concentration of PNA0, the frequency of non-hybridized PNAinduced blockade events which is proportional to (PNA0[𝑃𝑁𝐴:𝐷𝑁𝐴]𝑒𝑞.), would differ to a larger extent from the PNAinduced blockade events measured in absence of hybridization (proportional to PNA0), and thus increase the likelihood of accurate ssDNA detection present at low concentrations. So far in our study, we found that the optimal PNA concentration enabling ssDNA detection in symmetrical salts present in cis and trans chambers was 5 µM, resulting in a reliable sensitivity limit of the cis-added ssDNA of 0.05 µM (Fig. 1). A way to optimally detect PNA-induced blockades with PNA present initially at lower concentrations (< 5 µM), may call for increased transmembrane potential values; however, in such experiments we noted an increased mechanical instability of the bilayer membrane system, precluding reliable and long-lasting experiments. As an alternative solution to increase the throughput of PNAnanopore interactions while PNA is present with initially lower concentration values, without the need to re-engineer the analyte itself or the nanopore, or increase the transmembrane potential, we resorted to using asymmetrical salt concentrations (see ‘Simplified model for the transmembrane voltage- and salt gradient-dependence capture of a charged analyte inside a

Fig. 3 A salt gradient across the nanopore facilitates detection of PNA depletion following hybridization with nM concentrations from the complementary ssDNA. (I) With a symmetrical KCl buffer present across the nanopore and an applied transmembrane potential ΔV = -160 mV, as low as 3 µM from the cis-added polycationic peptide-functionalized PNAs, enables rare, but yet visible capture events (a, b) at the nanopore’s vestibule entry. With a 3 M KCl (trans):0.1 M KCl (cis) gradient maintained across the nanopore and a similar applied ΔV as above, cis-added polycationic peptidefunctionalized PNAs can be detected at bulk concentrations as low as 0.5 µM (c, d). (II) Cis-side, incremental application from the complementary cDNA in the nM range depletes the PNA’s bulk concentration via hybridization (a), and this phenomenon is seen as progressive increase in the association time (τon) of


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the PNA (c-f). The of τon vs. [cDNA] analysis (b) reveals that cis-side addition of cDNA at a concentration as low as 5 nM, can be detected via changes entailed in the association time (τon) of the PNA. In b, the dotted lines represent the 95 % confidence intervals for the average τon values (upward triangles), and the corresponding digits reflect the events number entering the statistics. This kinetic observation makes sense on the framework discussed above, as cDNA-PNA hybridization leads to a decrease in the free PNA concentration, which in turn reflects as a lower PNA-nanopore association rate, and correspondingly larger τon values. Detection experiments in a salt gradient as described above, enables sensing of cDNA with an approximately one order of magnitude increase in the sensitivity of detection, from approximately 50 nM in the case of symmetrical salts, to approximately 5 nM in the case of 3 M KCl (trans):0.1 M KCl (cis) gradient (Fig. 3, II, b). Real-time, single-molecule rupture of PNA-DNA duplexes containing distinct base mismatches Having established the detection capability of PNA-DNA duplexes containing various base mismatch degrees, our next objective was to assess the ability of the technique to probing PNA-DNA duplexes hybridization energy vs. mismatch length. To this end, we sought to capture and resolve in real-time, unimolecular PNA-DNA unzipping kinetics, and then analyze the stochastic unzipping events of duplexes containing distinct mismatches to derive values of the corresponding hybridization energy. We first established that the polycationic peptide-functionalized PNA-DNA duplex can optimally unzipped at the wild-type αHL’s β-barrel entry. By applying + ΔV’s across the nanopore, the ensuing electric field will guide the polycationic peptide tail from a PNA-DNA duplex into the vicinity of the α-HL’s βbarrel entry (Fig. 4), where the duplex will be further stabilized through the electrostatic attractive β-barrel entry – polycationic tail interactions, since it is known that electrostatic interactions are important for analyte detection and transport through the nanopores and membranes.73,81-83 Because the duplex cannot fit through the α-HL’s β-barrel domain (~ 2 nm in diameter), a captured PNA-DNA duplex will protrude the β-barrel with the polycationic tail alone, as the electric force stemming from the applied + ΔV threads this segment inside the nanopore. The negatively charged nucleotides from the captured PNA-DNA duplex will remain exposed to the trans chamber and sense an oppositely oriented electric force from the +ΔV, as compared to the polycationic peptide tail. It is the combination of these oppositely oriented forces acting on the captured molecule, that leads to the efficient duplex unzipping, reflected by the representative trace shown in Fig. 4, a-c.

Fig. 4 Irreversible duplex unzipping and quantitative determination of the PNA-DNA hybridization energy. Typical current traces measured at ΔV = + 200 mV, showing the real-time unzip on the trans side of the nanopore of PNA-cDNA (molar ratio of 1:5) (a), PNA-nDNA1_5 (molar ratio of 1:5) (b) and PNAnDNA3_5 (molar ratio of 1:5) duplexes (c). We indicate the duplex time to unzip (𝜏2𝑜𝑓𝑓) and passage time of the unzipped, polycationic peptide-functionalized PNA strand across the nanopore (𝜏3𝑜𝑓𝑓). The representative current signatures recorded at a lower transmembrane potential ΔV = + 160 mV, indicate that instead of unzipping, capture and subsequent release (𝜏1𝑜𝑓𝑓) of stable PNAcDNA (d), PNA-nDNA1_5 (e) and PNA-nDNA3_5 (f) duplexes occur more likely. Statistical analysis of average time value (𝜏𝑜𝑓𝑓) constructed from 𝜏2𝑜𝑓𝑓 and 𝜏1𝑜𝑓𝑓 time intervals recorded at distinct ΔVs, allowed the estimation of unzip (hybridization) energies of PNA-cDNA (g), PNA-nDNA1_5 (h) and PNA-nDNA3_5 (i) duplexes (see also main text). The vertical lines in panels g-i indicate the ΔV values assigned to the peak on the 𝜏𝑜𝑓𝑓 vs. ΔV dependencies, suggestive of a threshold (ΔVthreshold) beyond which unzip events become prevalent (see also text). As suggested in panels a-c, and evidenced previously, free polycationicfunctionalized PNAs translocate across the nanopore under the experimental conditions presented.65

As the polycationic segment from a hybridized PNA-DNA threads fully the nanopore’s β-barrel, it hinders the subsequent entry of the duplex inside the nanopore, due to topological exclusion. Assuming approximately 0.38 nm per amino acid on the extended peptide segment, the 10-mer arginine tail would span about 3.8 nm, namely 76 % from the β-barrel length. In this scenario, the polypeptide tail of the captured duplex does not encounter the approximately 1.4 nm in diameter constriction of the nanopore, where the maximum extend of ionic current obstruction is expected to occur,84,85 and hence the reduced degree of blockade (ΔIduplex, Fig. S3, d) as compared to the case when a single-stranded PNA segment was driven all the way across the nanopore through constriction region (ΔIPNA, Fig. S3, b). From this configuration the duplex can only move backwardly to the trans chamber (Fig. S3, d), or unzip under



the shear electric force sensed at the extremities of the duplex (vide supra), followed by the electrophoretic-driven transport of the PNA segment across the nanopore and ssDNA fragment back into the trans chamber (Fig. S3, e). Volumetric data support this interpretation, whereby the relative blockade amplitudes of the nanopore by the PNA molecule added alone in the trans chamber and that resulting from an unzipped DNAPNA duplex are practically similar (Table S1). During similar experimental conditions as above, negative ΔVs applied across the nanopore precluded duplex capture and unzipping at the nanopore’s β-barrel entry (Fig. S4). This is somewhat paradoxical, given that the net negative charge on the duplex and applied electric field, would guide the molecules toward the nanopore’s entry. We suggest that the cumulative effect of repulsive electrostatic interactions manifested between the negative moiety from the dipolar-like PNA-DNA duplex and the negatively charged β-barrel entry, on one hand, and the cis-to-trans oriented electric force acting on the polycationic tail from the PNA strand, on the other hand, result in a low likelihood capture probability of the duplex by the nanopore. Nanopore-based unzipping of individual PNA-DNA duplexes reveals single-base pair resolution mismatches To date and to mention just very few, elaborate models were proposed to extract kinetic information from forcespectroscopy experiments, as implicated by the generalization of the Kramers theory, particularly for the cases of time-varying force acting on molecular complexes.86,87 In simplified experimental paradigms however, when the electrical force acting on the duplexes is constant, combined with the fact that duplex formation and unzipping is an all-or-nothing process that occurs over a single, rate-limiting barrier, the classical Kramers’ rate model proved successful.49,51,65 By the use of a model previously proposed to describe the PNADNA unzipping inside the α-HL,65 (see also Fig. S5), we extracted the dwell times corresponding instances when the nanopore-captured PNA−DNA duplex returns to the trans side without unzipping (𝜏1𝑜𝑓𝑓; Fig. 4, f) and those indicative of the duplex unzip (𝜏2𝑜𝑓𝑓; Fig. 4, c), and the average time value (𝜏𝑜𝑓𝑓) was constructed from these numbers. The ΔV values corresponding the peak on the 𝜏𝑜𝑓𝑓 vs. ΔV for all ssDNAs tested (Fig. 4, g-i) were viewed as a threshold in the transmembrane potential (ΔVthreshold) beyond which unzip events on a captured duplex become prevalent. That is, for transmembrane potentials greater or equal to ΔVthreshold, the average 𝜏𝑜𝑓𝑓 constructed as indicated above, consist largely of 𝜏2𝑜𝑓𝑓 values, which quantify the time-to-unzip of a duplex, which shortens with an increase in the ΔV. To the opposite, for transmembrane potentials less than ΔVthreshold, the average 𝜏𝑜𝑓𝑓 consist largely of 𝜏1𝑜𝑓𝑓 values, which quantify the release of a captured duplex back to the trans side, against the electrophoretic force, which lengthens with an increase in the ΔV. This interpretation is supported by the experimental findings, namely ΔVthreshold (PNA-nDNA3_5) (~ 187 mV) < ΔVthreshold (PNA-nDNA1_5) (~ 200 mV) ≈ ΔVthreshold (PNA-cDNA) (~ 200 mV) (Fig. 4, g-i), which states essentially, that for less stable PNA-ssDNA duplexes with three unmatched bases, the electric force needed to break apart the dimer is lower as compared to more stable duplexes with perfect base match between strands (PNA-cDNA), or a single mismatch (PNA-nDNA1_5).

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As described in65 and applied to the model presented by Fig. S5, the average 𝜏𝑜𝑓𝑓 was correlated to the rate constants associated with the duplex unzip (𝑘𝐶𝑈) and duplex dissociation from 1

nanopore without unzip (𝑘𝐶𝑂) (𝜏𝑜𝑓𝑓 = 𝑘𝐶𝑂 + 𝑘𝐶𝑈), and the analytical description of these rate constants vs. ΔV write as 𝑘𝐶𝑈 (∆𝑉) = 𝑐𝑡1𝑒𝑥𝑝(𝑎∆𝑉2) and 𝑘𝐶𝑂(∆𝑉) = 𝑐𝑡2𝑒𝑥𝑝( ―𝑏∆𝑉2). The ct1 and ct2 constants represent values of the rate constants associated to the unzip and duplex dissociation, respectively, at V = 0, while a and b sum up contributions from all terms entering the values of rate constants written within the Kramers’ rate model.65 From the non-linear fit of data presented in Fig. 4, g-i, with an analytical function for (𝜏𝑜𝑓𝑓) constructed as above, the most relevant parameter derived was ct1 which, within the working model adopted herein, it writes: 𝑐𝑡1 =

𝑘𝐵𝑇 ℎ

(

𝑒𝑥𝑝 ―

∗ ∆𝐺0,𝐶𝑈

𝑘𝐵𝑇

) (s ). -1

𝑘𝐵𝑇

Replacing in this expression the Eyring frequency factor ( ℎ , where h is the Planck constant, kB and T represent the Boltzmann constant and absolute temperature) with 109 s-1,88 at a room temperature of T=300 K, the standard free energy barrier values for the duplex unzipping in the absence of the applied ∗ ) for all probe ssDNA sequences tested, were potential (∆𝐺0,𝐶𝑈 ∗ calculated as: ∆𝐺0,𝐶𝑈 (PNA-cDNA) = 86.8 ± 4.3 𝑘𝐽 𝑚𝑜𝑙𝑒 ―1, ∗ ∗ ∆𝐺0,𝐶𝑈 (PNA-nDNA1_5) = 65.6 ± 4.8 𝑘𝐽 𝑚𝑜𝑙𝑒 ―1 and ∆𝐺0,𝐶𝑈 ―1 (PNA-nDNA3_5) = 64.6 ± 3.5 𝑘𝐽 𝑚𝑜𝑙𝑒 , respectively. Within the statistical uncertainties, these results based on at least three independent experiments demonstrate that the probe (ssDNA)-target (PNA) mismatch degree affects the unzip energy barrier, and the discrimination reliability of the method ∗ reaches single-nucleotide selectivity (compare ∆𝐺0,𝐶𝑈 (PNA∗ cDNA) and ∆𝐺0,𝐶𝑈 (PNA-nDNA1_5) values). Conclusion and Outlook Although nanopore- and hybridization probes-based methods for sensing nucleic acids in solution were previously reported, a major limitation of the approach arises when short (< 30 bp) nucleic acids detection is needed, due to the fast translocation (order of tens of µs) of sequences across nanopores.69,89 Here, we demonstrate that wild-type α-HL nanopore in conjunction with polycationic peptides-conjugated PNAs, can be used to detect ssDNAs, without chemical modification, tagging, amplification, or adsorption to various surfaces, and examine hybridization energies of PNA-DNA duplexes in solution. By comparing similar duplexes with subtle changes in their degree of complementarity, we revealed that both the unzipping timescales are longer and hybridization energies larger for the perfect complement. In a cautionary tone, we stress that when considering the protocols described herein for probing real-life samples, preceding steps may be needed in order to minimize the likelihood of false positive signals, caused by non-specific binding of other proteins or molecular aggregates, nucleic acids, etc. to the polycationic peptidesconjugated PNAs. For example, preliminary filtration or purification procedures of the tested sample, in order to achieve an increased degree of selective detection of the nucleic acid probes, may be required.68,90


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We expect this method could be used as a powerful, cheap, easy-to-use and real-time exploration of short DNAs profiling, as well as for the analysis of other highly fragmented DNA samples, such as ancient DNA, or for the facile identification of single-base mismatches. Supporting Information The Supporting Information contains information regarding reagents and chemicals, additional experimental data and theoretical descriptions related to concepts presented herein.

Corresponding Author *[email protected] and [email protected]

Author Contributions The manuscript was written through contributions of all authors. / All authors have given approval to the final version of the manuscript. / #These authors contributed equally.

Acknowledgement 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, PN-IIIP1-1.1-TE-2016-0508 and PN-III-P1-1.1-PD-20160737 and 34PFE/19.10.2018.

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Nanopore-Assisted, Sequence-Specific Detection and Single

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