Nanoscale Probing of Informational Polymers with Nanopores

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Nanoscale Probing of Informational Polymers with Nanopores. Applications to Amyloidogenic Fragments, Peptides, and DNA−PNA Hybrids Tudor Luchian,*,† Yoonkyung Park,*,‡ Alina Asandei,§ Irina Schiopu,§ Loredana Mereuta,† and Aurelia Apetrei† †

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

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‡

CONSPECTUS: The decades long advances in nanotechnology, biomolecular sciences, and protein engineering ushered the introduction of groundbreaking technologies devoted to understanding how matter behaves at single particle level. Arguably, one of the simplest in concept is the nanopore-based paradigm, with deep roots in what is originally known as the Coulter counter, resistive-pulse technique. Historically, a nanopore system comprising the oligomeric protein generated by Staphylococcus aureus toxin α-hemolysin (α-HL) was first applied to detecting polynucleotides, as revealed in 1996 by John J. Kasianowicz, Eric Brandin, Daniel Branton, and David W. Deamer, in the Proceedings of the National Academy of Sciences. Nowadays, a wide variety of other solid-state or protein-based nanopores have emerged as efficient tools for stochastic sensing of analytes as small as single metal ions, handling single molecules, or real-time, label-free probing of chemical reactions at single-molecule level. In this Account, we demonstrate the usefulness of the α-HL nanopore on probing metal-induced folding of peptides, and to investigating the reversible binding of various metals to physiologically relevant amyloid fragments. The widely recognized Achilles heel of the approach, is the relatively short dwell time of the analytes inside the nanopore. This hinders the collection of sufficient data required to infer statistically meaningful conclusions about the physical or chemical state of the studied analyte. To mitigate this, various approaches were successfully applied in particular experiments, including but not restricted to altering physical parameters of the aqueous solution, downsizing the nanopore geometry, the controlled tuning of the balance between the electrostatic and electro-osmotic forces, coating nanopores with a fluid lipid bilayer, employing a pressure-voltage biased pore. From our perspective, in this Account, we will present two strategies aimed at controlling the analyte passage across the α-HL. First, we will reveal how the electroosmotic flow can be harnessed to control residence time, direction, and the sequence of spatiotemporal dynamics of a single peptide along the nanopore. This also allows one to identify the mesoscopic trajectory of a peptide exiting the nanopore through either the vestibule or β-barrel moiety. Second, we lay out the principles of an approach dubbed “nanopore tweezing”, enabling simultaneous capture rate increase and escape rate decrease of a peptide from the α-HL, with the applied voltage. At its core, this method requires the creation of an electrical dipole on the peptide under study, via engineering positive and negative amino acid residues at the two ends of the peptide. Concise applications of this approach are being demonstrated, as in proof-of-concept experiments we probed the primary structure exploration of polypeptides, via discrimination between selected neutral amino acid residues. Another useful venue provided by the nanopores is represented by single-molecule force experiments on captured analytes continued...

Received: November 11, 2018

© XXXX American Chemical Society

A

DOI: 10.1021/acs.accounts.8b00565 Acc. Chem. Res. XXXX, XXX, XXX−XXX

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Figure 1. (a) Reversible interactions of an analyte with a nanopore isolated in a lipid membrane, subjected to a potential difference (ΔV), are seen as stepwise blockades of the ionic current through the nanopore (Iopen) to a lower level (Iblocked). Analysis of ΔI = Iblocked − Iopen, blockade duration (τoff) and interevent intervals (τon), probe: (b) nanomechanics on individual biomolecules; (c) biopolymers (nucleic acids, peptides) sequence; (d) molecular recognition and single-molecule chemistry; (e) single peptide/protein (un)folding.

inside the nanopore, which proved useful in exploring force-induced rupture of nucleic acids duplexes, hairpins, or various nucleic acids-ligand conjugates. We will show that when applied to oppositely charged, polypeptide-functionalized PNA−DNA duplexes, the nanopore tweezing introduces a new generation of force-spectroscopy nanopore-based platforms, facilitating unzipping of a captured duplex and enabling the duplex hybridization energy estimation.

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INTRODUCTION The synergy of digital and nanofabrication technologies combined with life sciences advances, made possible through a convergent approach of otherwise well-delimited disciplines such as biology, physics, chemistry, and engineering, ushered the advent of the Fourth Industrial Revolution. On its wake, it holds the promise of technological leaps in genomics, proteomics, synthetic biology, and molecular engineering, all leading to “game-changing” technologies with considerable impact to society. Advances in nanotechnology and protein engineering paved the way for the advent of nanopore-based investigation of matter at a molecular level, which is nothing short of revolutionary in many areas and disciplines, and demonstrated the potential for applications ranging from sensing at the molecular level, the so-called “stochastic sensing”, of polymers, small molecules, enantiomers,1,2 pathogens,3,4 single-molecule chemistry investigation,5 RNA and DNA detection and analysis,6−11 peptide folding analysis,12−14 peptide sensing and identification,15−17 or to investigate the nanomechanics associated with (un)folding and unzipping of nucleic acids or nucleic acids−protein complexes.18−22 Riding on the current sophistication of technologies dedicated to single molecule detection, either protein- or solid-state-based nanopores have emerged as widely used,

powerful tools of investigation. Among the most commonly used protein-based nanopores systems are (i) α- and γhemolysin, from Staphylococcus aureus;23,24 (ii) MspA (Mycobacterium smegmatis porin A);25 (iii) the Phi29 connector channel;26 (iv) aerolysin, from Aeromonas hydrophila;27 (v) cytolysin A from Salmonella typhi (ClyA).28 Solid state nanopores have been fabricated using a wide variety of substrates, including Si3N4, Al2O3, TiO2, MoS2, HfO2, or graphene.29,30 The “right” nanopore for a specific task is dictated by the fundamental requirements, including sensitivity and specificity. The classical example is the real-time DNA sequencing with single-nucleotide resolution. The underlying hypothesis is that, as a single-stranded DNA journeys across the nanopore, its nucleotides will distinctly alter the ionic current through the nanopore, revealing through statistical analysis the DNA sequence. Ideally, the system should allow volumetric detection with single-base resolution, so that the four bases presented successively inside the nanopore would give rise to four distinct ionic current blockade patterns. From a historical perspective, the homoheptameric α-Hemolysin (α-HL) nanopore was first introduced to this task.6 The deciphered crystal structure of the protein revealed that the α-HL’s sensing zone (the ∼5 nm long β-barrel), is much longer then the base-to-base distance of DNA B

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Figure 2. (I) Traces reflecting α-HL-human Aβ(1−16) interactions in the absence (a) and presence of trans-added Cu2+ (b−d), at ΔV = −100 mV. The peptide-induced blockades are shown as upward spikes. The all-points histograms (e−h) illustrate the blockade level (#) induced by the metal-free peptide (Aβ) on the open α-HL current (*), and those corresponding to Cu2+-complexed peptide (Aβ-Cu2+) occluding the nanopore ($ and &). (II) Cu2+ dependence of average time measured between consecutive blockades caused by either a Cu2+-free or a Cu2+-complexed peptide (τON) (a), and the average duration of blockades (b) characterizing Aβ(1−16)−α-HL interactions. (III) Three-states Markov model describing the α-HL interactions with a metal-free (Aβ) or Cu2+-complexed peptide (Aβ-Cu2+). (Adapted with permission from ref 41. Copyright 2013 American Chemical Society.)

(∼3.4 Å). Even more challenging, α-HL has several recognition sites for nucleotides, so that clever protein engineering was necessary to identify all DNA bases.31 As an alternative, the single constriction of the octameric MspA (diameter of ∼1 nm and length of ∼0.5 nm) was proposed to yield better current signatures for each nucleotide of a ssDNA driven across it.32 For the same task, two-dimensional material (e.g., graphene, molybdenum disulfide) based nanopores are promising alternatives for single-nucleotide detection, due to their sub-nanometer thickness, that imparts unrivaled spatial resolution.33 In this Account, we overview our findings devoted to probing the interactions between metal cations and truncated amyloid peptides, which may evolve to the next generation of ultrasensitive single-molecule applications, enabling the fast characterization of the interplay between other analytes or metals and protein/peptides (e.g., α-synuclein, prion protein) misfolding, aggregation or conformational changes. We also describe recent paradigms to overcome one of the major obstacles for making nanopore-based analysis mainstream, namely the short residence time of analytes in the nanopore. It is also our aim to show how these may help the development of improved systems for structure recognition of peptides, or be useful for studying nanomechanics of individual molecules, realtime molecular detection and probing of ssDNA fragments.

(Iblocked) (Figure 1a). Three parameters provide microscopic insights into the analyte under study (Figure 1b−e): (i) the blockade amplitude ΔI = Iblocked − Iopen) (ii) blockade duration (τoff), and (iii) interevents time between successive capture events (τon). The first parameter provides structure-related information about the analyte,34 whereas the latter two, treated within the stochastically Markov series of events35 or the firstpassage time for one-dimensional diffusion,36 give more on the identity and concentration of analytes. It is accepted that amyloid-β (Aβ) peptides undergo a progressive aggregation from disordered peptides to insoluble fibers, leading to senile plaques and accumulation of neurofibrillary tangles in the Alzheimer’s (AD) patients’ brain,37 and that certain metal ions including Cu2+, Zn2+, and Fe3+ are important neurochemical factors promoting the Aβ conformational changes.38 Knowing that α-HL is a versatile tool to probe Aβ aggregation39 and Cu2+-triggered, peptides conformational changes,40 we used the truncated, more soluble Aβ(1−16) isoforms from human and rat to examine Aβ-Cu2+ interactions.41 When added on the trans side of a lipid membrane containing a single, cis(grounded)-added nanopore, trans-negative potentials drive the slightly anionic human Aβ(1−16) (charge ∼ −1.7 e− at neutral pH) into the nanopore’s lumen, leading to reversible blockades (Figure 2Ia). Trans side addition of Cu2+ elicits distinct types of Aβinduced blockades in the ionic current distribution (denoted by #, $, and &, Figure 2Ib−h), and the corresponding kinetic vs Cu2+ concentration is shown in Figure 2IIa and b. The histograms of blockades recorded in Cu2+-free buffer revealed a single peak corresponding to the Aβ−α-HL interactions (#; Figure 2Ia, e), while a second prevalent blockade appeared in the presence of Cu 2+ , suggestive of the (Aβ-Cu 2+ )−α-HL interactions ($; Figure 2, Ib−h) (Table 1). In excess of Cu2+, the metal-free, Aβ(1−16)-induced events (#) vanish, as the

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α-HL NANOPORE AS A LABEL-FREE PLATFORM FOR EXPLORING AMYLOID PEPTIDE−METAL INTERACTIONS Simply viewed, the analyte−α-HL interactions are seen as the nanopore switching between two conductive states, caused by the electrolyte dislodgement inside the nanopore by a residing analyte molecule, namely, the “open” state (Iopen, the analyte-free nanopore) and the “blocked” state of lower conductance C

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trations of trans-added Cu2+.41 We proposed that Cu2+-induced spatial conformations and the ensuing physical changes on such fragments is dependent on critical residues from the peptide.

Table 1. Blockade Amplitudes (ΔI) between Various Substates (#, *, &, $) and Percentage Blockade (%) Relative to Iopen, Associated with Human Aβ(1-16)−α-HL Interactions at ΔV = −100 mV, in the Absence and Presence of Cu2+a [Cu ] (μM)

ΔI (pA)

% blockade

0 25

139.33 ± 2.78 (#−*) 129.83 ± 0.84 (#−*) 141.65 ± 1.01 ($−*) 146.19 ± 0.12 (#−*) 161.07 ± 0.1 ($−*) 113.79 ± 0.94 (&−*) 148.00 ± 1.70 ($−*) 99.03 ± 5.95 (&−*)

89.21 87.70 95.67 88.49 97.50 68.88 95.46 63.87

2+

100

200

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ELECTROOSMOTIC CONTROL OF THE MOLECULAR TRANSPORT ACROSS THE α-HL One of the pitfalls of the nanopore-based analyte recognition is the high translocation speed of analyte, which hampers its accurate identification and analysis.35 To alleviate this, several strategies were devised and reviewed previously.44 We exploited the interplay between electrophoresis, stemming from electric forces acting on the analyte from the applied ΔV, and electroosmosis, arising from the directional, net water flow through ion-selective nanochannels,45 to modulate the passage of a peptide (CAMA6-P6; KWKLFKKIGIGKFLQSAKKFNH2) across the α-HL.46 The two glycines (G) at positions 9 and 11 separated by isoleucine (I) at position 10 promote a kinked hairpin-like state, rendering the peptide more prone to electroosmotic flow influence. At neutral pH, positive ΔV’s guide the trans-added peptides (charge of ∼ +8e−) toward the α-HL’s β-barrel, and the subsequent capture and translocation were seen as B1 events (Figure 3Ia, d). Although less prominent, positive ΔV;s lead concomitantly to the cis-to-trans oriented motion of water across the slightly anionic selective α-HL (PK+/PCl−= 0.86 at pH 7.1), opposite to the peptide movement through electrophoresis. The drift velocity of a peptide along the nanopore is the vector sum of the electrophoretic (velectrophoretic; trans-to-cis oriented, in our case) and electroosmotic (velectroosmotic; cis-totrans oriented) components, which under simplifying assumptions (e.g., geometrically homogeneous nanopores) write46

a

Reprinted with permission from ref 41. Copyright 2013 American Chemical Society.

blockades seen ($) reflect solely the α-HL−Cu2+-complexed Aβ(1−16) interactions (Figure 2Id, h). Due to their low amplitude and fast kinetics, &-like events were assigned to transient collisions of the Aβ-Cu2+ with the nanopore; their scarcity (∼5% from the total blockade events in Figure 2, I h) motivated exclusion of the corresponding substate from the scheme shown in Figure 2III and subsequent analysis. For analysis, we knew that Cu2+ coordination to the Aβ leads to its structural modification, and Aβ(1−16)-Cu2+ stoichiometry is 1:1.42 Within the scheme shown in Figure 2III, the analysis of the lumped association time intervals (τON) corresponding to a “Cu2+-free” or a “Cu2+-complexed” Aβ interacting with the nanopore helped estimate the dissociation constant of Cu2+ to Aβ(1−16) (Kd = 4.5 × 10−7 M).40 Similarly, we demonstrated that Zn2+ and Fe3+ were less effective in binding to the human Aβ(1−16).43 Cu2+ complexation by the rat Aβ(1−16) lacking the His-13 residue involved in metal coordination, resulted in a higher frequency of blockades reflecting the peptide-α-HL interactions at increasing concen-

vdrift = velectrophoretic − velectroosmotic =μ

(P − PK +) ΔV NhI /(|e−|Spore[H 2O]) − Cl − lpore (PCl − + PK +)

(1)

Figure 3. (I) Traces reflecting the trans-added, CAMA-P6-α-HL interactions at ΔV = +50 mV, in a 2 M KCl buffer at pH = 7.1 (a), pH = 4.5 (b), and pH = 3.3 (c). The zoomed-in panels display the open-pore currents across the nanopore in the absence of peptide partitioning (“O”), whereas the blockade substates “B1” and “B2” visible in acidic buffers illustrate a peptide residing temporarily in the α-HL’s β-barrel and vestibule, respectively. In panels (d)−(f), we represent the scatter plots of dwell time vs relative blockade amplitude of “B1” and “B2” blockade events, from the open pore “O”; for clarity, we encircled the B1 state at neutral pH (d) and the B2 state at pH = 4.5 (e). (II) Voltage-dependence of the rate of peptide exiting the B2 substate (rateoffB2). (III) Probing the distinct microstates accompanying the cis-added peptide passage across the α-HL at ΔV = −50 mV and pH = 4.5. A peptide imported into the vestibule (the O → B2 transition) can move reversibly between the vestibule and β-barrel (the B2 ↔ B1 transitions), before exiting the nanopore through β-barrel (the B1→ O transition (a), or it escapes to the cis side (the B2→ O transition) (b). (Adapted with permission from ref 46. Copyright 2014 authors.) D

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Figure 4. Ion current traces illustrating the trans-added, CAMA-P6−α-HL interactions at pH = 7 (I) and pH = 2.8 (II), at positive (Ia and IIa) or negative ΔV’s (Ib and IIb). At neutral (Ia, c) or acidic pH (IIa, d), positive ΔV’s enable trans-added CAMA-P6−nanopore interactions, under the electrophoretic force (Felp). Unlike the neutral buffer (Ib, d), at pH = 2.8 peptides partition into the α-HL’s β-barrel at negative potentials as well (upwardly oriented blockade events) (IIb, e), driven by the enhanced electroosmotic flow across the α-HL (Felo). (Adapted with permission from ref 48. Copyright 2016 American Chemical Society.)

with Spore as the average cross-sectional area of the α-HL, ΔV is the applied potential, μ is the electrophoretic mobility of the peptide, Nh is the number of waters associated with each mobile ion, [H2O] is the water concentration, I is the ionic current recorded while a peptide resides inside the nanopore, e− is the electronic charge, PK+ and PCl− are the ionic permeabilities of the nanopore, and lpore is the nanopore’s length. The pH-dependent selectivity of the α-HL provides a facile opportunity to influence the peptide translocation. At lower pH values, the α-HL increases its anion selectivity (PK+/PCl− = 0.44 at pH 4.4), leading to a corresponding increase in the electroosmotic velocity of water, whereas the electrical charge on the peptide and velectrophoretic remain unchanged. Consequently, a reduction of the net drift velocity of the peptide was anticipated. As seen in Figure 3Ib−f, at low pHs, a second blockade substate (B2) becomes visible. From volume-exclusion arguments (α-HL’s β-barrel inner diameter is ∼20 Å, and its vestibule diameter is ∼46 Å), we attributed the blockade B1 to the peptide residing in the β-barrel, and the blockade B2 to the peptide located inside the nanopore’s vestibule. Thus, the electroosmotic-mediated reduction of peptide’s velocity allowed to visualize the translocation process as it proceeds sequentially through the nanopore’s β-barrel and vestibule domains. This is in nice agreement with previous work, in which authors have employed Langevin dynamics simulations to predict the emergence of distinct ionic current blockade changes accompanying polymers translocation across the α-HL.47 By modeling the peptide residence inside the vestibule within the framework of the first passage time for one-dimensional diffusion of a charged particle in a constant electric field, we

estimated the probability density function of peptide’s sojourn times in the vestibule, at pH = 4.5. By nonlinear fitting of data in Figure 3II with the theoretically derived rateoff B2 vs ΔV dependency,46 we calculated the peptide’s diffusion coefficient inside the vestibule D = 1.5 × 10−12 m2 s−1. The 2 orders of magnitude lower value of D than of a similar peptide dissolved in water, reflects the restricted movement of the peptide within a nanoscopic space. With the trans-added peptide, level B1 always preceded level B2 (Figure 3Ib, c), before the peptide dissociates from the nanopore (level O), thus revealing the directionality of peptide translocation. The interaction between the cis side added peptide and the α-HL, resulted in the emergence of the B2 substate (peptide in the α-HL’s vestibule) always preceding the B1 substate (peptide inside the β-barrel) (Figure 3III). Single-molecule electrophysiology traces revealed that O substate appeared after B1 substate or B2 substate, thus capturing for the first time the escape directionality of the initially vestibule-trapped peptide, namely, to the trans (O appears after B1) (Figure 3, III a) or cis side (O appears after B2) of the membrane (Figure 3IIIb).

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ELECTROOSMOTIC FLOW-MEDIATED PEPTIDE CAPTURE BY THE NANOPORE

We suggested that, by working with even more acidic buffers than in the preceding chapter, the electroosmotic force exerted on the CAMA-P6 may become as large as to dominate the electrophoretic transport. Indeed, experiments carried out at pH = 2.8 have shown that electroosmotic flow enables peptide capture by the α-HL nanopore against the electrophoretic force.48 E

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Figure 5. (I) Traces showing the reversible α-HL−CP2a interactions, with the trans-added peptide at ΔV = +70 mV (a), ΔV = +90 mV (b), and ΔV = +100 mV (c). The scatter plot distribution of current blockade vs interevents and blockade durations show that as ΔV increases, a concomitant decrease of the interevents time intervals (τon) and an increase of the blockade-events durations (τoff) ensues. The quantification of these parameters in −1 + terms of association (rateon = τ̅−1 on ) and dissociation rates (rateoff = τ̅off ) is represented in (II). A positive ΔV acts on the positively charged end (Npore) of − − the peptide (|F+|) and drives into the nanopore, while an opposite force (|F |) acts on the negatively charged segment (Npore) of the peptide (III and IV). When the two forces, |F+| and |F−|, balance (zero net force stage, IVa), the peptide achieves a metastable state (IV b). (Adapted with permission from ref 44. Copyright 2015 authors.)

Figure 6. (I) In its metastable state, the middle section of the peptide (“X”) fluctuates near the α-HL’s constriction region, generating current blockades suggestive of its structure. (II) Current recordings measured at ΔV = +70 mV showing the pore blockades induced by three peptides (Ac− (R)12−(A)6−(E)12−NH2) (a), (Ac−(R)12−(W)6−(E)12−NH2) (b), or (Ac−(R)12−(A)3−(W)3−(E)12−NH2) (c), added in the trans side at pH = 7, and the corresponding all-points histograms illustrating the distribution of the sub-blockade events generated by groups of at least three A or W residues, marked with & ( ΔIblocked = 0.83 ± 6 × 10−3) and $ ( ΔIblocked = 0.93 ± 4 × 10−3), distinct from the fully blocked substate (#) ( ΔIblocked = 0.95 ± 6 × Iopen

Iopen

Iopen

10−3 in a or ΔIblocked = 0.98 ± 9.1 × 10−3 in b). (Adapted with permission from ref 50. Copyright 2017 American Chemical Society.) Iopen

Positive ΔV’s determine CAMA-P6−nanopore reversible interactions, regardless of the pH value (Figure 4I a, c and IIa, d). At negative ΔV’s, such interactions are absent, as the electrophoretic force (Felp) drives the peptides away from the nanopore (Figure 4Ib, d). Interestingly, at pH = 2.8, peptides enter the protein β-barrel even at negative ΔV’s, against the outwardly oriented Felp (Figure 4IIb, e). This indicates the manifestation of the electroosmosis counterflow (Felo) enabling the change of the peptide’s drift motion direction, otherwise

ruled by the sign of the electrophoretic force. In addition, the oppositely acting electroosmotic and electrophoretic forces at pH = 2.8 and negative ΔV’s, slow down the peptide trafficking across the nanopore, enough as to identify the peptide location inside the nanopore and movement directionality. As above, the volumetric interpretation of data shown in Figure 4IIc are consistent with a scenario in which the initially, β-barrel entrapped peptide (generating IL) moves to the vestibule (substate IV), followed by release−recapture events of the F

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Figure 7. (I) Representative blockades associated with the PNA-DNA capture inside the nanopore, namely, lower amplitude events of duration τ1off (a and c) and events displaying an initially lower blockade step of duration τ2off (b), assigned to a captured PNA−DNA, followed irreversibly by a slightly larger blockade step of duration τ3off (b), indicative of the PNA−DNA unzip (d). (II) Voltage-dependence of the average dwell time τ̅off measured on a population of time intervals comprising τ1off (unsuccessful unzips) and τ2off events (successful unzips). Data points were fitted according to the model (III). (Reprinted with permission from ref 53. Copyright 2017 American Chemical Society.)

combination of the applied electric field and the charges at the ends of the peptide.

peptide to and from the cis side (IO ↔ IV transitions), before it ultimately escapes on the cis side (IV → IO transition). This allowed us to quantify a thermodynamic model with activation barriers, describing the free energy changes of a peptide moving along the α-HL, between its inner nanovolumes.48

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THE NANOPORE-TWEEZER FACILITATES MOLECULAR RECOGNITION ON THE PRIMARY SEQUENCE OF PEPTIDES Previously, the nanopore technology was proposed to offer useful structural information on peptides.49 To reveal sequence information on peptides with the nanopore-tweezer, we engineered peptides to contain either six A, six W, or combination of three A and three W residues (the ‘X’ denoted region), flanked by oppositely charged tails at neutral pH, containing 12 R or E residues (Figure 6). From geometric considerations, when a metastable peptide resides inside the αHL through the mechanism detailed above, (F⃗ elp(R12 tail) ≈ F⃗ elp (E12 tail)), the nanopore’s constriction region is populated mainly by the peptide’s middle domain residues (Figure 6I). Knowing that the α-HL’s constriction region (length ∼ 0.6 nm) may harbor ∼1.6 amino acids at a time, we posited that the current fluctuations associated with the movement of the metastable peptide, leading to substates with statistically significant relative blockades ( ΔIblocked ) (& and $ in Figure 6II),

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NANOPORE-TWEEZER PARADIGM TO SLOW DOWN ANALYTE TRANSLOCATION ACROSS THE α-HL In a distinct approach leading to the “nanopore-tweezer” technique,44 we achieved retarded peptide motion across the α-HL, through oppositely oriented, electric forces acting from the ΔV on peptides termed CP2a (Ac−(E)12−(N)12−(R)12− NH2), whose C- and N-termini contained basic and acidic amino acids (Figure 5I and II). At positive ΔV’s, trans-added peptides driven in the vicinity of the α-HL’s β-barrel by thermal motion get captured with the positively charged moiety head on (Figure 5III). During translocation, the peptide’s negatively charged moiety experiences an oppositely oriented force, directed toward the trans side. An electrostatic “tug of war” between the charges on opposite sides of the peptide and the ΔV occurs. At some point during movement, the net force acting on the molecule decreases to virtually zero, with the peptide being trapped in a metastable state close to the middle of the nanopore (Figure 5IV).44 We developed a mathematical model for the free-energy profile along the translocation pathway, and found that increasingly ΔV values led to correspondingly lower rateoff values for the peptide, as shown in Figure 5, II. We found that the free-energy barrier for peptide to escape from the nanopore increases with the electric field E.44 This paradigm provides effective means to improve the signal-to-noise ration of single molecule nanopore-based measurements. The “zero net force” state of the peptide does ends and the peptide release occurs, when thermal fluctuations experienced by it are large enough to overcome the barrier generated by the

Iopen

reflect the volumes of at least three residues from the peptide’s middle section “X”, thus facilitating their detection.50

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DISSECTING PEPTIDE NUCLEIC ACID−DNA DUPLEXES WITH A NANOPORE TWEEZER As before, we reasoned that by applying the nanopore tweezer to an asymmetrically charged molecular duplex, not only detection is improved in terms of sensitivity and selectivity,51,52 but a more efficient duplex dissociation may result, through the combination of forces acting oppositely on its termini. We employed a cationic polypeptide-functionalized peptide nucleic acid (PNA) (Ac−(R)9−5′-GTGATATACG-3′)hybridized with a complementary ssDNA strand (the underlined sequence matches the G

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PNA; 5′-CCCCCCCCGTATATCAC-3′), and demonstrated that electric forces from the applied transmembrane potential, acting oppositely at both ends of the PNA−DNA duplex trapped inside the α-HL, facilitate unzipping.53 The blockade events corresponding to the PNA-DNA-α-HL interactions, can be ascribed to the duplex partially entering the α-HL’s β-barrel with the PNA-attached cationic polypeptide head on, and then retracting back to the trans side (“1”-like events in Figure 7Ia), or instances when following entrance inside the nanopore (“2”like events in Figure 7Ib), duplex unzipping occurs, facilitating the electrophoretic-mediated translocation of the unzipped PNA monomer across the nanopore (events denoted by “3” in Figure 7Ib). Within the Markov theory of transitions between discrete states,53 assuming that the electric charge (qeff) from the PNA− DNA sensing the electric field while plugging the nanopore depends linearly vs ΔV (qeff ≈ ctΔV) (Figure 7III), and knowing that according to the model shown in Figure 7III, the relationship between the average value of lumped blockade intervals represented by τ1off and τ2off (τ̅off) and the corresponding 1 rate constants for dissociation is τoff ̅ = k + k , and the average 1O

ORCID

Tudor Luchian: 0000-0002-9388-7266 Funding

The work was supported by National Research Foundation of Korea (NRF) (2016R1A2A1A05005440), Global Research Laboratory (GRL) (NRF-2014K1A1A2064460), Institute for Information & Communications Technology Promotion (IITP) Grant (MSIT) (2017-0-01714), and Grants PN-IIIP4-ID-PCE-2016-0026, PN-IIIP1-1.1-TE-2016-0508, and PNIII-P1-1.1-PD-2016-0737. Notes

The authors declare no competing financial interest. Biographies Tudor Luchian is professor in the Department of Physics, at the ‘Alexandru I. Cuza’ University in Iasi, Romania, where he works on nanoscale biophysics. He obtained his Ph.D. in Physics in 1997 from “Karl-Franzens” University of Graz, Austria, where he studied GIRK ion channels with electrophysiology techniques.

1U

standard molar energy for unzipping an individual base-pair was evaluated at 1.17 kcal mol−1.

Yoonkyung Park is with the Department of Biomedical Science and Research Center for Proteineous Materials, Chosun University, South Korea, where she works on antimicrobial peptides. She obtained his Ph.D. in Molecular Biology in 1999 from Chosun University of Kwangju City, South Korea, where she studied interleukin-2 protein production in plant materials.

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CONCLUDING REMARKS Despite recent advances, bottlenecks in the development of nanopore-based instruments still exist, limited mainly by spatial resolution provided by nanopores, sampling frequency, and high trafficking rates across the nanopore. Low-noise CMOS microelectronics enabling current measurements through nanopores at bandwidths beyond 1 MHz, combined with (i) rich alternatives for controlling analytes capture rates and translocation speed, (ii) successful efforts to develop sub-nanopores, and (iii) protocols enabling parallel readout of multiple nanopores and development of deep learning algorithms facilitating prediction-making based on single-molecule ionic current fluctuations, are expected to have a strong impact on point-of-care personalized biomedical diagnostics, proteomics, and single-molecule manipulation. The nanopore approach described herein to investigate metals−Aβ peptide interactions may complement existing spectroscopic techniques (e.g., CD, ThT fluorescence) to study more effectively structure−function relationships of various Aβ oligomerization and misfolding inhibitors, and screen drugs (e.g., β-sheet breaker peptides, antibodies) counteracting the Aβ toxicity. Within the nanoporetweezer paradigm, future developments enabling unidirectional movement of polypeptides across the nanopore may lead to refined approaches useful to probing the presence of specific domains or mutations, post-translational modifications, and ultimately achieve peptide sequencing at a unimolecular level. When applied to DNA/RNA/PNA molecules, the nanopore tweezer approach introduces a new generation of forcespectroscopy platform, enabling simultaneous, real-time nucleic acids detection via complementary hybridization and hybridization energy assessment. This may prove useful for diagnostic and accurate genotyping at the single-molecule level, or other nanotechnological applications enabling quantification of forces exerted between biomolecules.

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Alina Asandei is senior research scientist at the Interdisciplinary Research Institute, ‘Alexandru I. Cuza’ University in Iasi, Romania, where she works on nanopore-based stochastic sensing. She obtained her Ph.D. in Physics in 2009 from “Alexandru Ioan Cuza” University of Iasi, where she studied antibiotic biomolecules-biomimetic lipid systems interactions. Schiopu Irina is research scientist at the Interdisciplinary Research Institute, ‘Alexandru I. Cuza’ University in Iasi, Romania. Her research interests revolve around single-molecule studies of membrane-active peptides, dendrimers, nucleic acids. She received her Ph.D. in Physics in 2014 from ‘Alexandru I. Cuza’ University in Iasi, where she studied amyloid peptides−lipoproteic systems interactions. Loredana Mereuta is associate professor at the Department of Physics, ‘Alexandru I. Cuza’ University in Iasi, Romania, where she works on single-molecule investigation of peptides- nanopores interactions. She completed her Ph. D. in Physics in 2010, at the ‘Al. I. Cuza’ University of Iasi, with a thesis about antimicrobial peptides-lipid membrane interactions. Aurelia Apetrei is lecturer at the Department of Physics, ‘Alexandru I. Cuza’ University in Iasi, Romania, activating in the field of molecular biophysics. She obtained her Ph.D. in Physics from the same institution in 2011, on mechanisms of membrane disruption by membrane-active peptides. Her activity is focused on peptide-lipid membrane interactions and unimolecular characterization of analytes with nanopore-based techniques.

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