Electron Transfer in Spacer-Free DNA Duplexes Tethered to Gold via

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Electron Transfer in Spacer-Free DNA Duplexes Tethered to Gold via dA Tags Rui Campos, László Kékedy-Nagy, Zhe She, Rana Sodhi, Heinz-Bernhard Kraatz, and Elena E Ferapontova Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b01412 • Publication Date (Web): 25 Jun 2018 Downloaded from http://pubs.acs.org on June 25, 2018

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Electron Transfer in Spacer-Free DNA Duplexes Tethered to Gold via dA10 Tags Rui Campos,† László Kékedy-Nagy,† Zhe She,‡ Rana Sodhi, ‖ Heinz-Bernhard Kraatz‡‖ and Elena E. Ferapontova†* †

Interdisciplinary Nanoscience Center (iNANO), Science and Technology, Aarhus University, Gustav Wieds Vej 14, 8000 Aarhus C, Denmark

‡

Department of Physical and Environmental Sciences, 1095 Military Trail, University of Toronto Scarborough, Toronto, Ontario M1C 1A4, Canada ‖

Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, Ontario M5S 3E5, Canada Corresponding’s author e-mail: [email protected]

KEYWORDS: DNA, Electron Transfer, Gold, Methylene blue, Polyadenine tag, Phosphorothioated polyadenine tag;

ABSTRACT. Electrical properties of DNA critically depend on the way DNA molecules are integrated within the electronics, particularly on DNA-electrode immobilization strategies. Here, we show that the rate of electron transport in DNA duplexes spacer-free tethered to gold via the adenosine terminal region (a dA10 tag) is enhanced compared to the hitherto reported DNA-metal electrode tethering chemistries. The rate of DNA-mediated electron transfer (ET) between the

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electrode and methylene blue intercalated into the dA10-tagged DNA duplex approached 361 s-1 at a ca. half-monolayer DNA surface coverage ΓDNA (with a linear regression limit of 670 s-1 at ΓDNA→0), being 2.7-fold enhanced compared to phosphorothioated dA*5 tethering (6-fold for the C6-alkanethiol linker representing an additional ET barrier). XPS evidenced dA10 binding to the Au surface via the purine N, while dA*5 predominantly coordinated to the surface via sulfur atoms of phosphothioates. The latter apparently induces the DNA strand twist in the point of surface attachment affecting the local DNA conformation and, as a result, decreasing ET rates through the duplex. Thus, spacer-free DNA coupling to electrodes via dA10 tags thus allows a perspective design of DNA electronic circuits and sensors with advanced electronic properties and no implication from more expensive, synthetic linkers.


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INTRODUCTION Numerous bioelectronic applications of DNA molecules strongly depend on the way DNA selfassemblies are integrated with the electronic devices, which electronic properties are preconditioned by the mode of DNA binding to electrodes.1-8 The appropriate strategies for the DNA-electrode surface coupling can be efficiently assessed by electrochemical analysis of the DNA-mediated electron transfer (ET) rates in DNA duplexes tethered to electrodes via a variety of surface chemistries9-11 offering a practically beneficial alternative to spectroscopic12-14 and conductivity analysis.15, 16 The most widely used approach for DNA attachment to gold surfaces is via either C3 or C6 alkanethiol linkers introduced into one end of the DNA sequence by automated synthesis providing stable and reproducible binding DNA to gold.1,

17-19

Thin films of thiol-modified

oligonucleotides on gold have been exploited extensively for monitoring the hybridization of complementary oligonucleotide strands, interactions of small molecules with DNA duplexes, and identification of single nucleotide mismatches.10, 20, 21 However, alkane spacers introduce an additional barrier for ET,2 resulting in very moderate rates of ET between the electrode and redox species intercalated into the DNA duplex, as a rule ranging between 1 and 100 s-1,22,

23

far below photo-induced ET rates of 105 - 1010 s-1.24-26

Spacer-free DNA tethering to electrodes via adenosine phosphorothioate tags (dAx*) in which one of the non-bridging phosphate oxygens was replaced by sulfur (Figure 1) resulted in enhanced rates of DNA-mediated ET,8 though the observed 259 s–1 were still far below the theoretical predictions.2


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Figure 1. Schematic representation of electron transfer between the gold electrode and methylene blue, mediated by DNA duplexes attached to gold via a C6 alkanethiol linker and phosphorothioated polyadenosine dA*5 and polyadenosine dA10 tags in the 3´-terminal part of DNA.

We hypothesized that moderate ET rates in dA*x-tethered double stranded DNA resulted from “twisted” binding of DNA duplexes to gold via sugar-thiophosphate backbone of the dA*x tag (Figure 1). In order to further advance electronic communication between the electrodes and DNA-intercalated dye, we aimed at alternative spacer-free ways of DNA tethering to gold, such as via non-thiolated oligodioxynucleotides: dA and dG were shown to strongly interact with gold,17, 27-31 with binding energies of 31 – 35 kcal mol-1 31, 32 comparable to that of thiols (35 – 40 kcal mol-1).33 Adsorption of dA on gold through N3 or N7

34, 35

was strong enough for thiol

replacement.27 dAx tags seemed to be most attractive for DNA tethering to gold, both due to higher (electro)chemical stability (higher oxidation potentials of a nucleobase) and tag’s linear structure uncomplicated by intramolecular intercations.20 Used both as anchor and surface blockage agents, poly-adenosine (polyA) tags provided a better control over DNA orientation, conformation and hybridization on gold.30, 36 PolyA tails with five or more dA adsorbed strongly on gold nanoparticles (AuNPs) in a fairly uniform fashion,37 with cations playing a significant role in forming a tightly packed DNA film.38 DNA tethering to AuNPs through polyA not only


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improved stability of binding but also hybridization ability due to controllable spatial separation between strands.39, 40 However, no analysis of electrical properties of dA-tagged DNA has been reported. Here, in order to achieve DNA binding to gold in the most natural “untwisted” way and yet have a spacer-free DNA attachment to the gold surface, we coupled DNA to Au via the 3´-located polyA10 tag composed of naturally occurring adenosine base. We electrochemically interrogated ET mediated by DNA tethered to Au electrodes via the dA10 and dA*5 tags and a C6-alkanthiol linker and correlated results with the surface attachment chemistries.

MATERIALS AND METHODS Materials. DNA sequences (from 5´ to 3´) dA10-DNA: GTT GTG CAG CGC CTC ACA AC AAA AAA AAA A; complementary DNA (cDNA): GTT GTG AGG CGC TGC ACA AC; SNP-containing cDNA: GTT GTG AGG CAC TGC ACA AC, and their analogues with dA*5 and HS-C6-linkers (Figure 1) were from Metabion Int., Martinsried, Germany. Components of buffer solutions, methylene blue (MB), Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), 1mercapto-hexanol and hexaammineruthenium (III) chloride were from Sigma-Aldrich, Germany. All solutions were prepared with deionized Milli-Q water (18 M, Millipore, Bedford, MA, USA). Electrode modification with DNA. Prior to modification, gold electrodes (CH Instruments, Austin, Texas, USA; diameter 2 mm) were first electrochemically cleaned in de-aerated 0.5 M NaOH by cycling potential between -0.4 and -1.6 V (0.05 V s-1, 10 cycles), then mechanically polished on a microcloth pad using 1 μm diamond and 0.1 μm alumina slurries (both from Struers, Copenhagen, Denmark), washed with Milli-Q water and ultrasonicated in a 1:1


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ethanol:water solution for 10 min. Then the electrodes were electrochemically polished in 1 M H2SO4 and 1 M H2SO4/10 mM KCl. The electrochemically active surface area of the electrodes was estimated from peaks corresponding to the gold surface oxide reduction in the last voltammogram recorded in 0.1 M H2SO4. Before DNA immobilization, the electrodes were kept in absolute ethanol for 30 min. DNA was immobilized by overnight incubation of the electrodes in a 10 μM dsDNA solution (1 h hybridization of 10 μM linker/tag-modified sequences with 15 μM cDNA in 10 mM Na-phosphate buffer solution, PBS, pH 7, containing 50 mM NaCl and 0.1 M MgCl2), as described elsewhere,41 a C6-linker being also treated with TCEP for di-sulfide bond reduction. The dsDNA-modified electrodes were thoroughly rinsed with 10 mM PBS, pH 7.0, and either incubated in 2 mM 1-mercapto-hexanol solution in the same buffer for 30 min and then used (C6- and dA*5-tags) or immediately used in electrochemical experiments/kept at 4 °C in the same buffer. The DNA surface coverage is referred to the electrochemically active electrode surface area of 0.057±0.005 cm2 determined from the gold surface oxide reduction peaks in 0.1 M H2SO4.42 In SNP studies, the electrodes modified with fully complementary dsDNA were “de-hybridized” by thorough washing with water and after that overnight “rehybridized” by reaction with 15 μM SNP-containing cDNA. Instrumentation and Procedure. Cyclic voltammetry (CV) and square wave voltammetry (SWV) were performed in a dark-glass three-electrode cell connected to the potentiostat μAUTOLAB type III (Eco Chemie B. V., Utrecht, Netherlands) equipped with GPES 4.9 and with NOVA 1.11 software. An Ag/AgCl (3 M KCl) electrode was the reference electrode and a platinum flag (1 cm2) was the auxiliary electrode. The working solution was 10 mM PBS, pH 7, containing 0.1 μM MB. Working solutions were de-aerated by Ar for at least 15 min prior to data acquisition and blanketed under Ar during the experiments. The DNA surface coverage was


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determined with 0.2 mM Ru(NH3)63+ following the established procedure.43 CV was run within the 25 and 0.05 V s-1 range, and SWV was recorded at frequencies between 1000 and 8 Hz. Analysis of ET rate constants ks. The ks was determined from CV data by using the Laviron formalism44 for CV peak potential separations smaller than 200/n mV, from ks=(F/RT)(mnν), where the tabulated m values depend on the potential peak separation related to the number of electrons n involved in the ET reaction. All peak potentials were corrected for solution Ohmic drop45 iRs, where the solution resistance Rs of 1064±37 Ω was determined by electrochemical impedance spectroscopy. The ks was determined from SWV data46 from ks=ωmax fmax, where fmax is the critical frequency, at which the (I/f) relation of the measured current I to the frequency f at which it is maximal, and ωmax is the kinetic parameter depending both on α and nEsw (these values are tabulated). XPS measurements. Data were collected using a Thermofisher Scientific K-Alpha system (Thermofisher Scientific—E. Grinstead, UK) at the Ontario Centre for the Characterization of Advanced Materials (OCCAM) at the University of Toronto. A monochromatic Al Kα X-ray was used with a nominal spot size of 400 m. Au on Si samples were attached to the sample plate with clips and following survey spectra (pass energy (pe) - 200 eV), the spectral regions of interest were obtained at both low energy resolution (pe – 150 eV) for relative peak intensity, and at higher energy resolution for more chemical information. For the latter, the pass energy was set to 25 eV for the more intense peaks (C 1s, Au 4f and O 1s) and at 50 eV for the weaker peaks (N 1s and S 2p). The photoelectron take-off angle (ToA) was 90 relative to the surface. No charge compensation was applied and the energy scales were aligned to place the Au 4f7/2 at 84.0 eV. All data processing was performed using the software supplied with the system (Avantage 5.926). Relative atomic % composition was obtained from the individual low-


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resolution spectra after subtraction of a Shirley type background47 and application of the supplied sensitivity factors (20.735, 2.881, 1.881, 1.676 and 1 for Au 4f, O 1s, S 2p, N 1s and C 1s respectively). Curve-fitting was also performed on the high-resolution peaks using the same software. The run was repeated but this time using a ToA of 20 relative to the surface (i.e. more surface-sensitive). This was accomplished by placing the samples using double-sided C tape on a wedge angled 20 from the plate normal.

RESULTS AND DISCUSSION Kinetic analysis of ET. ET between gold electrodes and MB intercalated into DNA duplexes tethered to gold via either dA10 and dA*5 tags or an alkanethiol linker was studied by CV with electrodes modified with fully complementary DNA duplexes. In all cases, a couple of redox peaks from MB could be followed, with a formal redox potentials of –225 ± 6 mV (the C6 linker), –235 ± 11 mV (dA*5) and –291±6 mV (dA10), shifting to more negative values with the increasing negative charge on the tether (Figure 2), consistent with a negative potential drop in the electric double layer that might affect the MB binding affinity and thus the apparent potential.48 The CV peak currents linearly depended on the potential scan rate (Figure S1, ESI) consistent with the surface-confined electrochemical process49 and in agreement with electrochemistry of MB well-coupled to the DNA base pair π-stack.23 It is worth to mention that additional blocking of the electrode surface with 1-mercapto-hexanol resulted in a better developed electrochemistry of MB intercalated into the DNA-dA*5 tagged duplex and in less discrepancy of the data, consistent with previous reports,8 while in the case of the dA10 tag, as followed both from Ru hexamine analysis of the DNA surface coverage and MB electrochemistry, DNA was essentially removed from the electrode surface (Figure S2). Thus,


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the stability of dA10 binding to macroscopic gold electrode surface was less than shown with AuNPs,37-40 but sufficient for ET and surface-binding studies. (NB: For longer than overnight immobilization times (24-48 h) stability of dA10 binding to gold essentially increased; however, for comparative purposes, the routine DNA immobilization protocols were used in the present work).

Figure 2. (A) Schematic representation of electron transfer between the gold electrode and methylene blue (MB), mediated by the DNA duplex attached to gold via either polyadenosine dA10 or phosphorothioated polyadenosine dA*5 tags in the 3´-terminal part of DNA. The dA10 tag interacts with Au predominantly via N7 (alternatively, N3); (B) Representative background-corrected CVs, recorded for dA10 tagged (solid line), dA*5 tagged (dashed blue line) and C6 (dashed green line) DNA-modified gold electrodes in 10 mM PBS, pH 7, in the presence of 0.1 μM MB, normalized for the MB surface coverage Γ (pmol cm–2), scan rate v (5 V s-1), and electrode area A (cm2).

Heterogeneous ET rate constants, ks, evaluated for ET in dA10-tetheterd DNA both by SWV 50, 51 and by CV analysis44 (Figures S3, S4) gave very similar values approaching 350-360 s-1 for perfectly matched duplex at its around half-compact monolayer coverage,22 which dropped 40±2% when the duplex had an A→C mismatch in the 10th base pair position (Figure 3, Table 1). The SNP-sensitivity of ET rate confirmed that ET was mediated by the DNA duplex indeed.


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Therewith, in high ionic strength solutions (0.15 M NaCl), screening the charge on DNA molecules and preventing electrostatic and groove binding of MB,52 the ET rates for the dA10tagged DNA duplex decreased only 7%, with this also excluding the possibility that measured ET rates were related to MB electrostatically adsorbed on the dA10 tails on the electrode surface.

Figure 3. Dependence of the normalized relation (i/f) between the SWV peak currents i and the frequency f in semi-logarithmic coordinates recorded with the gold electrodes modified with (solid line) fully complementary double-stranded DNA-dA10 and (dashed line) with a SNP-containing DNA-dA10 duplex. Inset: Normalized as in Fig. 2 representative background-corrected CVs, same notations as in the main figure.

For comparable surface coverages, the rate of the DNA-mediated ET in dA10-tethered DNA was three to four-fold higher than ET in DNA tethered to gold via the phosphorothioated dA*5 tag (Figures 2B, S5, Table 1). It was 6-fold higher than ET in DNA duplexes immobilized on gold through the C6 alkanethiol linker, consistent with previous reports on dominating contribution of electron tunneling through the linker to the overall DNA-mediated ET rate.2, 8 Thus, the observed


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increase in the efficiency of ET through the dA10-tethered DNA duplex should relate to the way that DNA binding to gold occurred. Table 1. DNA surface coverage, ΓDNA, and the ET rate constant, ks, for perfectly matched (PM) and SNP-containing DNA duplexes tethered to gold via dA10 and dA*5 tags and C6 alkanethiol. ΓDNA/ pmol cm-2

ks/s-1 (CV)

ks/s-1 (SWV)

ks0/s-1 (ΓDNA→0)

ks0/s-1 (ΓDNA→max)

PM

5.5±0.2

350±25

361±12

670±29

223±10

SNP

5.2±0.2

191±5

213±11

n.d.

n.d.

dA*5

PM

3.7±0.2

93±6

132±7

332±27

132±7

C6

PM

5.3±0.3

59±7

60±5

263±30

61±4

Tether used

dA10

XPS studies. The experimental XPS studies were carried out on dA*5 and dA10 on polycrystalline Au on Si (for XPS survey scan see Figures S6-S8). The N 1s region of dA*5 and of dA10 adsorbed on Au in Figure 4 shows signals stemming from polyA adsorption on the Au surface, at 399.2 eV and 400.2 eV due to the purine N, and at higher binding energy at 401.6 eV due to the exocyclic amino N, consistent with the XPS signals reported for dA15/dA20 strands adsorbed on gold exhibited by the purine N and exocyclic N.53-55 This latter signal remained unchanged in both systems as it was not expected to reflect surface interactions.34 Purine signals for phosphothioated dA*5 are observed at 399.4 and 400.5 eV. What is noticeable is the significant difference between the signal intensities of the purine N for dA*5 and for dA10 (Figure 4). While for dA*5 the signal at 399.4 eV accounted for 55.5% of the purine N signal, the signal at 400.5 eV made up the remainder. For dA10, the signal at higher binding energy dominated, with the intensities of the two signals becoming 26% and 74%, respectively. Given that the structural dissimilarity between two systems relates to the presence of S in the phosphothioate


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backbone of dA*5, we attribute these differences to two different binding modes of dA*5 and dA10 to gold.

Figure 4. Representative XPS spectrum of (A) dA*5 and (B) dA10 on Au/Si substrates showing the N 1s region. Experimental data is shown in black and the fitting is shown in pink. Deconvolution of the data allows identification of three distinct N signals represented in blue, green and red, assigned to (blue) the amino group, (green) Au-coordinated purine N, and (red) free uncoordinated purine N. Specific signals are observed for dA*5: 401.4, 400.5, and 399.4 eV and for dA10: 401.6, 400.2, and 399.2 eV.

While it was recognized before that the adenine nucleobase alone had multiple potential coordination sites for surface interactions in multiple ways and in different orientations,56, 57 the presence of the carbohydrate in the nucleoside introduces steric factors that alter the coordination of the adenosine nucleoside dA compared to the isolated nucleobase A.31 Detailed SERS and IR studies of adenosine monophosphate (AMP) adsorption in gold suggested coordination of AMP’s N7 (73.5 kcal mol-1) and N3 (79.5 kcal mol-1) but not the amino NH2 group (64.7 kcal mol-1) that has been calculated to have a significantly weaker binding energies with Au+.34 More recent DFT calculations support AMP’s adsorption on Au(100) surfaces via N7,58 in agreement with SERS and XPS analysis of adenine on Au.54 To form a film on Au surfaces, dA10 has to rely solely on interactions of the purine N with the Au surface (Figure 2A). Our studies would


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suggest that for dA10 two populations of the purine N exist, one that is bound to the Au surface accounts for 74% of the purine N and a minor component of the presumably uncoordinated purine N (Figure 4B). This was in sharp contrast to dA*5 binding: In addition to purine N-coordination, S-coordination to the Au surface via phosphothioate was possible and that gave rise to two populations of the purine N in a ratio of about 1:1 (55.5 to 44.5) (Figure 4A). With this, the signal from Aucoordinated purine N reduced, due to Au-N interactions which were significantly weaker compared to Au-S interactions increasing the fraction of dA5* S-coordinated to gold (Figure S9). The Au-S interactions show the XPS signal at 161.8 eV. Given the broadness of sulfur XPS signals in general (Figure S9), we are only able to provide an approximate ratio of bound/uncoordinated S of about 4:1. The binding energy of adsorbed S on Au(111) was reported by Vericat and coworkers and S2p is close to 161 eV. 59 We also want to make it clear that we are not interpreting the XPS data in terms of surface concentrations. Similarly stronger coordination of phosphorothioated polyA* (and thiolated polyA) and higher stability of its binding over polyA was observed on AuNPs, suggesting even exclusive coordination of the tags through thiophosphates.60 Consistent with that, our electrochemistry data can be interpreted in a way that additional blocking of the electrode surface with 1-mercaptohexanol left on the gold surface only the dA*5-tagged DNA bound to the electrodes via Au-S chemistry. In the case of the dA10 tag, 1-mercapto-hexanol then removed almost all dA10-tagged DNA molecules attached to gold via much weaker Au-N coordination binding. Thus, in contrast to phosphorothioated dA*5 bound to gold via the S-modified phosphate group, consistent with previous reports,34,

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the dA10 tag binding occurred via the DNA base itself


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indeed. Such difference in the DNA attachment to the conductive support obviously provided more intimate contact between DNA base pair π-stack and the electrode surface, resulting in a more efficient electrode coupling and, as a result, faster DNA-mediated ET reaction. Dependence of ET rates on the DNA surface coverage. The rates of DNA-associated ET were shown to significantly depend on the DNA surface coverage.8, 61 To get more universal kinetic data, variation of ET rates was studied over a wider range of the DNA surface coverage. Consistent with previous reports,8,

61

the increasing DNA surface coverage resulted in the

decreasing ET rates for any type of DNA tethering (Figure 5), approaching the limiting values shown for compact monolayers of C6-tethered DNA2, 23 already at DNA surface coverage ΓDNA exceeding 50% of a theoretical monolayer22 (Figure 5, ΓDNA > 4 pmol cm-2). The tendency of the ks leveling at ΓDNA>3 pmol cm-2 was also observed in the mixed DNA-dA*5/1-mercaptohexanol SAM, but not in DNA-dA10 SAMs, for which a continuous drop in the ET rates with increasing ΓDNA was followed (Figure 5).

Figure 5. Dependence of the rate of ET, ks, on the DNA surface coverage, ΓDNA, for dA10 (black circles and line), dA*5 (blue squares and line) and C6 alkanethiol (green triangles and line).


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Similar dependences were earlier observed for ET proceeding both in Ru(NH3)63+-wires formed along the DNA duplex61 and via the DNA duplex, from the electrode to the DNA-intercalated MB.8 Such DNA-packing dependent decrease in the ks correlates with the increasing complexity of interactions between the redox probe and the DNA duplex, and for the earlier studied hexadecylpyridinium-bis-chloranilato-antimonyl (V) hexamine complex it was addressed by varying interactions either between the adsorbed molecules or between the molecules and the electrode.62 In DNA duplexes vertically aligned in the electric field,41,

63

it may be rather

connected with the variations in the anisotropic polarizability of DNA molecules in thin DNA films of different densities.22 To obtain the limiting ks values, ks0, we extrapolated ET rates (linear regression analysis) to DNA surface coverages approaching 0 (ΓDNA→0) and to maximal DNA surface coverages (non-linear sigmoidal regression fitting) approaching a theoretical limit22 of ca. 9 pmol cm-2 (ΓDNA→max). It is worth to mention that despite the larger footprint of the dA10 tether essentially high ΓdA10-DNA could be interrogated, due to the absence of 1-mercapto-hexanol treatment removing weakly (NAu) bound DNA molecules. For ΓDNA→0, the C6 linker provided the lowest ks0 of 263 s-1, with dA*5 and dA10 approaching 333 s-1 and 670 s-1, correspondingly; quite similar relations between the ks0 were obtained for ΓDNA→max (Table 1). Consistent with previous reports,8 the linker-free DNA attachment allowed more efficient electrical communication between the electrode and DNA-intercalated MB, with a dA*5 tag superior to the commonly used C6-linker. The dA10 tag further improved electrical coupling between the DNA duplex and the electrodes, with an impressive 2-fold increase in the ET rate of DNA-mediated ET. Thus, the mode of DNA attachment to the conductive support appears to critically affect the DNA-mediated transport of electrons to the DNA-intercalated species, most probably through the electronic coupling


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between the metal electrode and the π-stacked base pairs of the DNA double helices. In our case, binding between N3/N7 of a dA base next to the one forming the duplex and gold apparently provided the improved coupling between the rest of the dsDNA base pair π-stack and metal surface. Similar effects are expected for other DNA-intercalating redox species, whose redox transformations occur within the same potential window (at negative electrode potentials), since both DNA surface orientation, sub-molecular organization and mobility of bases strongly depend on polarization applied.41 Along with that, despite the fast ET rates, DNA-dA10 mediated ET does not yet reach efficiency of the photo-excited ET,24-26, 64 being at least 100 times slower than the lowest limits shown in solution. We can dispute that this (electrochemical) way measured ET rates may not reflect the inherent ET properties of DNA duplexes, since the electric field applied not only induces the ET reaction but also strongly affects the conformation (A- versus B-form) and sub-molecular structure of gold-tethered DNA duplexes, including the π–stacking and mobility of the base pairs, by this destabilizing the double helix structure,41 which can even result in DNA melting not associated with the charged sugar-phosphate backbone of DNA.65 Induced changes then might result in a poorer than expected ET properties of the DNA duplex. 733 s-1 have been demonstrated in 16-mer DNA duplexes attached to the electrodes via the 5´-C4-linker, with a daunomycin intercalator covalently attached to the G-rich binding sites.2 Such a higher ET rate support our suggestion that the lower ET rates observed with a linker-free DNA-electrode modifications, among other things, may be connected with the effects the electric field produces on structural and electrical properties of DNA. Those effects may be most pronounced in the absence of a linker operating not only as an extra tunneling barrier but also as a kind of protective “cushion” against electric-field induced conformational changes.


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CONCLUSIONS Here, we showed that the linker-free attachment of DNA duplexes to gold electrodes via the dA10 tag allowed reaching the rates of the DNA-mediated electron transfer varying between 223 and 670 s-1 for compact (ΓDNA→max) and very diluted (ΓDNA→0) DNA monolayers, compared to 132 and 332 s-1 shown for the phosphorothioated dA*5 tag, correspondingly. The acceleration of ET in dA10-tagged DNA duplexes as compared to the linker-free attachment via the phosphorothioated dA*5 tag was connected with different ways of the tags binding to gold, for dA10 proceeding solely through the purine N. This way of the surface attachment provided the most efficient electrical coupling between the electrode and the ET-mediating DNA base-pair πstack. Though not reaching the rate limits shown with a photo-excited ET, DNA tethering to metal electrodes through the dA10-tag may be a compromising and beneficial alternative to the currently existing DNA-electrode surface attachment chemistries for DNA-mediated bioelectronics applications, both simplifying and cheapening the design of the DNA-based components.

ASSOCIATED CONTENT Supporting Information. Raw cyclic and square wave voltammetry data, Laviron’s plots; XPS survey scans and XPS spectra of dA*5 in the S 2p region. AUTHOR INFORMATION Corresponding Author Corresponding author, [email protected]

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Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT The work was supported by the NUMEN project funded by Danish Council for Independent Research (DFF-FTP-4005-00482B) and Danish National Research Foundation (DNRF81). Funding from NSERC (RGPIN-2016-06122) is greatly appreciated. ABBREVIATIONS CV, cyclic voltammetry; ET, electron transfer, dsDNA, double stranded DNA; MB, methylene blue; NP, nanoparticle; pe, pass energy; SWV, square wave voltammetry; SAM, self-assembled monolayer; ToA, photoelectron take-off angle. REFERENCES (1). Long, Y.-T.; Li, C.-Z.; Sutherland, T. C.; Chahma, M.; Lee, J. S.; Kraatz, H.-B. A comparison of electron-transfer rates of ferrocenoyl-linked DNA. J. Am. Chem. Soc. 2003, 125, 8724-8725. (2). Drummond, T. G.; Hill, M. G.; Barton, J. K. Electron transfer rates in DNA films as a function of tether length. J. Am. Chem. Soc. 2004, 126, 15010-15011. (3). Anne, A.; Demaille, C. Electron transport by molecular motion of redox-DNA strands: Unexpectedly slow rotational dynamics of 20-mer ds-DNA chains end-grafted onto surfaces via C(6) linkers. J. Am. Chem. Soc. 2008, 130, 9812-9823. (4). Mie, Y.; Kowata, K.; Kojima, N.; Komatsu, Y. Electrochemical properties of intrastrand cross-linked DNA duplexes labelled with Nile Blue. Langmuir 2012, 28, 17211-17216. (5). Farjami, E.; Campos, R.; Ferapontova, E. E. Effect of the DNA end of tethering to electrodes on electron transfer in methylene blue-labeled DNA duplexes. Langmuir 2012, 28, 16218-16226.


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Table of Contents Figure

10X


5X

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Electron Transfer in Spacer-Free DNA Duplexes Tethered to Gold via

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