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Electrostatic Cycling of Hybridization Using Nonionic DNA Mimics Sade Ruffin, Isabella A Hung, Ursula M Koniges, and Rastislav Levicky ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.7b00100 • Publication Date (Web): 15 Jun 2017 Downloaded from http://pubs.acs.org on June 19, 2017

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Electrostatic Cycling of Hybridization Using Nonionic DNA Mimics Sade Ruffin, Isabella A. Hung, Ursula M. Koniges and Rastislav Levicky* New York University Tandon School of Engineering, Dept. of Chemical and Biomolecular Engineering, 6 MetroTech Center, Brooklyn, NY 11201. *Corresponding author: [email protected]

ABSTRACT: This study demonstrates efficient electrostatic control of surface hybridization through use of morpholinos, a charge-neutral DNA mimic, as the immobilized "probes". In addition to being compatible with low ionic strengths, use of uncharged probes renders the field interaction specific to the nucleic acid analyte. In contrast to DNA probes, morpholino probes enable facile cycling between hybridized and dehybridized states within minutes. Impact of ionic strength and temperature on effectiveness of electrostatics to direct progress of hybridization is evaluated. Optimal electrostatic control is found when stability of probe-analyte duplexes is set so that electrostatics can efficiently switch between the forward (hybridization) and reverse (dehybridization) directions.

KEYWORDS: electrochemical, PNA, electrode, bioaffinity, assay, surface, monolayer

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The charged character of nucleic acids has naturally stimulated interest in using electric fields to control their behavior at interfaces for sensing and related applications. Electric fields can provide orientational control, allowing shifting between adsorbed and upright conformations.1–3 Such conformational switching was subsequently pursued for biosensing.4 Electric fields have been also proposed for direct control of hybridization between surface-bound "probe" and solution "target" strands.5–18 In these applications electric fields are intended to interact with the oligonucleotide charges to facilitate or hinder hybridization. Conde and co-workers reported that attractive biases of 1 V accelerated hybridization by nearly a billion fold, resulting in hybridization times of less than a msec.5,6 These authors considered that the fields facilitated hybridization of targets after they already adsorbed to the probe-modified support, rather than influence mass transport of targets to the probe layer. Using more modest biases of 0.2 V, Su et al observed a similar instantaneous like onset of hybridization after a "lag period" of no hybridization.8 In both these studies hybridization was determined from fluorescence signals after washing and drying of the DNA layers. In contrast, when hybridization was followed in-situ under attractive 0.3 V potentials, such strong acceleration was not observed.7 A number of studies also demonstrated that repulsive biases can assist the release of targets from probe-target duplexes.7–16 One explanation for dehybridization under repulsive biases is through electrostatic mechanisms,18 but other mechanisms were also put forth. Johnson et al observed dehybridization of duplexes formed by two complementary strands of peptide nucleic acids (PNA) when sufficiently negative (-0.8 V) potentials were used. Since PNAs are uncharged, electrostatics should not be responsible; rather, the authors suggested that electron transfer to the duplex pi stack caused its destabilization.19 The authors further suggested that


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such nonelectrostatic mechanisms could also explain denaturation of DNA-DNA duplexes under conditions of weak electrostatics, such as at high ionic strengths. More recent work considered the effects of dynamic fields on hybridization. Gebala and coworkers reported that application of potential cycles at 5 Hz under high ionic strengths (~ 1 mol L-1) approximately doubled the hybridization yield compared to applying a constant attractive potential.17 This interesting effect was not seen at lower (~ 0.02 mol L-1) ionic strengths. The physical mechanism was thought to involve structural dynamics of the DNA probe layer that facilitated hybridization when the layer organization was forced to constantly respond to switching fields. The primary motivation for using electric fields to modulate rates and equilibria of surface hybridization is convenience for diagnostic applications. For fully general capability it is desirable to accelerate as well as reverse hybridization at will. As noted above, use of DNA probes has led to inconsistent outcomes for attractive fields showing both extreme5,6,8 and little impact on forward rates,7 while achievement of fast (e.g. on a minute timescale) dehybridization has required assistance from buffer exchanges12,15 or clever use of charged nanoparticle tags to amplify electrostatic repulsions.14 Use of charged probes like DNA imposes certain limitations on electrostatic regulation of hybridization. One issue is that charged probes retain an excess of mobile counterions at the surface. This excess leads to enhanced local ionic strength, and thus to stronger screening of electric fields. In addition, charged probes can also respond to applied fields. Such responsiveness could interfere with hybridization; for example, attractive biases could cause the probes to adsorb to the solid support and block their binding to target strands. Lastly, hybridization to DNA probes requires that salt concentration be sufficiently high.20 This


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requirement limits optimization of electrostatic control by preventing evaluation of lower ionic strength conditions. These

considerations

motivated

exploration

of

uncharged

morpholino

(MO)

oligonucleotides as probes to facilitate electrostatic regulation of hybridization. In contrast to negatively charged DNA, morpholinos have a nonionic backbone consisting of alternating morpholino rings connected by neutral phosphorodiamidate linkages, Fig. 1A, and their hybridization with DNA is insensitive to salt conditions.21–23 Selection of morpholinos over other uncharged DNA analogues, such as PNA, was motivated by their good hybridization properties as well as synthetic flexibility with regard to length and base composition.22,24 The favorable properties of morpholinos have motivated a number of recent studies into their sensing applications.25–34 These applications have benefitted from the morpholinos’ ability to hybridize under low ionic strengths to, for example, destabilize interfering secondary structure in the sample25,26 or to improve sensitivity of charge-based transduction.30,35 DNA and MO probe monolayers of an identical 20mer base sequence (5' TTA AAT TCT GCA AGT GAT TT) were prepared on mercaptohexanol (MCH) passivated gold rotating disk electrodes. The first three thymine residues on the probes served as a linker to facilitate hybridization. Probe coverages were in the range of 1.5×1012 to 2×1012 cm-2, corresponding to a 7 to 8 nm average separation between probes. Thus modified electrodes were placed into 10 nmol L-1 solutions of complementary 17mer targets (5' TCA CTT GCA GAA TTT AA). Hybridization was monitored as a function of time while the potential applied to the electrode was stepped between various settings. At each potential, the extent of hybridization was determined every two minutes by calculating the surface coverage of targets from the charge Q


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needed to switch the oxidation state of their ferrocene labels. Q was calculated from fast cyclic voltammetry (CV) sweeps by integration of the corresponding peaks (Fig. S1). As shown in Fig. 1B, only slight if any effects on hybridization from applied fields were noticeable for either DNA or MO probe monolayers in 0.1 mol L-1 sodium phosphate buffer (SPB). Markedly better control was observed for MO probe layers if the SPB concentration was lowered to 0.01 mol L-1, with clearly delineated periods of acceleration under attractive (positive) potentials and halting under repulsive (negative) biases. The corresponding CV data are presented in Fig. 1C. DNA probes did not hybridize sufficiently in 0.01 mol L-1 SPB to allow quantification, as shown in Fig. S2, an observation attributed to destabilization of DNA-DNA duplexes at the low ionic strength. Based on these results subsequent experiments focused on MO probes and low salt conditions. The results for MO monolayers in Fig. 1B show that lower ionic strengths improved responsiveness of hybridization to applied potentials. This is attributed to further reach of surface electric fields at these salt concentrations. The electrostatic Debye screening length,36 rD, in 0.01 mol L-1 pH 7 SPB is about 2 nm; therefore, surface fields extend several nm into solution. As the length of a 17mer duplex is about 6 nm, this means that duplexes are substantially exposed to the field over most of their length. In comparison, longer probes would place more base pairs outside reach of the field, which would reduce the ability to regulate hybridization since a smaller fraction of duplex base pairs would be affected. A mismatch of probe size and extent of the fields can also be realized by increasing ionic strength; for example, in 0.1 mol L-1 buffer rD shrinks to 0.7 nm, resulting in field-duplex interaction over only a small portion of a duplex and a concomitant loss of control, as seen in Fig. 1B.


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The data in Fig. 1 did not demonstrate reversal of hybridization, only its halting. It would be useful to actuate both the forward and reverse reactions, so that the probe layer can be brought from a given initial state to a desired final state. This requires the field-induced perturbation to switch between conditions accelerating hybridization and those that reverse it. This requirement can be best met if duplex stability, in the absence of applied fields, is marginal. Then, electrostatics can be used to tilt the balance in favor of the forward or the reverse direction, stabilizing or denaturing probe-target duplexes as needed. Fig. 2A presents results of a temperature series for a fixed buffer strength of 0.01 mol L-1. These experiments used alternating 6 min rounds of attractive +0.3 and repulsive -0.3 V potential pulses. For 35 oC and below, MO-DNA duplexes remained sufficiently stable so that repulsive fields triggered only modest dehybridization within the allotted period. Increasing the temperature to 40 oC, however, lowered duplex stability sufficiently so that repulsive rounds clearly tipped the balance toward denaturation. To confirm that this reversal reflected changes in duplex stability, these experiments were repeated for a target with a central C/T mismatch (5' TCA CTT GCC GAA TTT AA; mismatch underlined). According to the proposed mechanism a duplex destabilized by a mismatch should display a lower threshold temperature for dehybridization. Indeed, the mismatch target exhibited hybridization switching already at 35 oC as shown in Fig. S3. Use of a partly matched target with just a six-nucleotide complementary region (5' ACT TGC ATC ATC ATA ACT CAC; complementary region is underlined) did not result in significant signal even after 16 minutes at +0.3 V (Fig. S4), verifying that the cycling involved sequence-specific hybridization. Fig. 2B contrasts the full match and mismatch data at 35 oC, illustrating the pronounced effect of the mismatch. The strong contrast in responses of the full match and mismatch


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sequences is potentially useful for controlling sequence stringency in applications. This control could be realized, for example, by using trains of potential pulses to accelerate replacement of mismatched by fully matched sequences during competitive hybridization. Development of such functionality will require optimization with respect to pulse amplitude, duration, mismatch position, and target length. Fig. S5 illustrates the expected variation of full match to mismatch signal with applied potential, as estimated from the noncompetitive hybridization results of Fig. 2B. Ultimately, electrostatic regulation of hybridization is constrained by magnitude of the available perturbation. The electrostatic energy Eel of an MO-DNA duplex interacting with an applied surface field was estimated by integrating the product of the local potential V(x), expressed relative to V = 0 in bulk solution, with the duplex charge

Eel =

−e V ( x)dx l duplex B

∫

(1)

layer

where x is measured normal to the electrode surface. In Eqn. 1, the duplex linear charge density e/lB has been set at one elementary charge -e per Bjerrum length37 lB, in accordance with counterion condensation theory.38 The Bjerrum length is lB = NAe2/(4πεrεoRT) where NA is Avogadro's number and εr and εo are the relative and vacuum permittivities; physically, lB represents the distance at which interaction between two elementary charges in a dielectric medium is comparable to thermal energy. In water at room temperature lB is approximately 0.7 nm. V(x) was obtained numerically from a Poisson-Boltzman calculation following the model in reference 39. For the calculation the MCH layer was assigned a dielectric constant of 4.0 and a thickness of 1.2 nm, as estimated from fits to capacitance of MCH monolayers following reference 35. The MO-DNA duplex layer was approximated as a slab with a dielectric constant


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that of water and a uniformly smeared density of negative charge as specified by the measured duplex coverage and charges per duplex L/lB ≈ 9, where L = 6 nm is the duplex length. The potential at the electrode/MCH interface was set to -0.25 V, representing the difference between -0.3 V used for electrostatic denaturation and the potential of zero charge (pzc) for morpholinomodified electrodes. The pzc was determined experimentally from the minimum in the double layer capacitance to be -0.05 V (Fig. S6). For simplicity in evaluating Eqn. 1 it was assumed that under repulsive biases duplexes orient perpendicularly to the surface. Other orientations could be included through Boltzmann partitioning based on electrostatic energy of a given tilt angle.2 The assumption of a normal orientation provides a lower limit on Eel. Fig. 3 shows the V(x) profile calculated for 0.01 mol L-1 phosphate buffer and typical duplex coverage of 5 × 1011 cm-2, with the hatched area representing the integration region over the 6 nm thickness of the duplex layer. The calculation yields Eel = 13 kJ mol-1, or about 5RT. This magnitude can be compared to an equivalent change in temperature. Using ∆Go = ∆Ho T∆So, treating ∆Ho and ∆So as independent of temperature, and taking ∆So for MO-DNA surface hybridization as about half its solution value23 (Supporting Information) leads to ∆So ≈ -0.5 kJ mol-1 K-1. A ∆Go change of 13 kJ mol-1 then corresponds to a temperature increase of 13 kJ mol1

/ 0.5 kJ mol-1 K-1 = 26 oK. Since this is approximately the width of an MO-DNA surface melting

transition,23 the prediction is that if the temperature is set just at the low side of a transition then applying a -0.3 V bias should be sufficient for achieving nearly complete denaturation. Such a prediction is in reasonable agreement with the trends in Fig. 2. Repeating the same calculation for 0.1 mol L-1 buffer leads to Eel = 1.4 kJ mol-1, or about 0.5RT. This low value, compared to thermal energy, agrees with Fig. 1B in that field effects were too weak to be significant in 0.1 mol L-1 buffer.


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The present study demonstrated facile switching between forward and reverse directions of surface hybridization when charge neutral DNA mimics were used as the probe molecules. At the lower ionic strengths found to be effective, the control mechanism was consistent with simple electrostatics in which surface charges on the electrode interact with backbone charges on the analyte to stabilize or destabilize its presence at the surface. Other mechanisms of electrochemically influencing duplex stability, such as electron transfer to the duplex pi stack19 or local perturbation of buffer pH as in certain commercial technologies,40 are not expected to be significant at the modest potentials used. The combination of biases gentle to biomolecular monolayers and rapid, within minutes, cycling of hybridization opens up opportunity for development of direct field effect protocols for controlling sequence stringency and other aspects of surface nucleic acid bioassays.

Acknowledgment This work was supported by the National Science Foundation (DMR 12-06754, CBET 1600584), and by New York University.

Supporting Information Description of experimental methods including analysis of CV data, hybridization to DNA probes at low ionic strengths, hybridization to mismatched targets, determination of the potential of zero charge, and characterization of hybridization thermodynamics in solution. This material is available free of charge via the Internet at http://pubs.acs.org.

Notes The authors declare no competing financial interest.


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Figure 1. (A) Molecular structure of the morpholino (MO) probe with detail of the end modifications. (B) Effect of applied potentials on hybridization of DNA targets to complementary DNA and MO probe layers. Background colors delineate 16 min periods of different applied potentials as indicated. Potentials are expressed relative to a Ag/AgCl/3 mol L-1 NaCl reference electrode. For clarity, the curve for DNA probes in 0.1 mol L-1 buffer was offset by multiplying it by 1.5. Experiments were carried out at room temperature, using 10 nmol L-1 target concentrations. (C) CV data for hybridization to MO probes in 0.01 mol L-1 buffer in part (B). Inset: A full CV trace. Main plot: Enlarged region corresponding to the red rectangle in the inset, showing progress of hybridization as a function of applied potential.


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Figure 2. (A) Hybridization of DNA targets to complementary MO probes under alternating attractive (0.3 V) and repulsive (-0.3 V) pulses, as a function of temperature. (B) Comparison of hybridization responses for full match targets to that for targets with a single central mismatch, at 35 oC. In all cases targets were present as a 10 nmol L-1 solution in 0.01 mol L-1 PBS, pH 7.


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Figure 3. Potential profile V(x) during a denaturation round, calculated for a 0.01 mol L-1 phosphate buffer and a MO-DNA duplex coverage of 5 × 1011 cm-2. The electrode/MCH interface is at x = 0. The potential is referenced relative to the potential of zero charge for morpholino-modified, MCH-passivated electrodes. Most of the potential drop occurs across the MCH passivation layer.


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