Modulation of Charge Transport Across Double-Stranded DNA by the

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Modulation of Charge Transport Across Double Stranded DNA by Site-Specific Incorporation of Copper bis-Phenanthroline Complexes Philippe Dauphin-Ducharme, Fiora Rosati, Andrea A. Greschner, A. Dowine De Bruijn, Danielle Salvatore, Violeta Toader, Kai Lin Lau, Janine Mauzeroll, and Hanadi F. Sleiman Langmuir, Just Accepted Manuscript • DOI: 10.1021/la504300g • Publication Date (Web): 15 Jan 2015 Downloaded from http://pubs.acs.org on January 23, 2015

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Modulation of Charge Transport Across Double

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Stranded DNA by Site-Specific Incorporation of

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Copper bis-Phenanthroline Complexes

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Philippe Dauphin-Ducharme‡, Fiora Rosati‡, Andrea Greschner, A. Dowine De Bruijn, Danielle

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Salvatore, Violeta Toader, Kai Lin Lau, Janine Mauzeroll* and Hanadi Sleiman*

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Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montreal, Quebec,

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H3A 0B8 (Canada)

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Keywords: DNA, Metal-DNA, Charge mediation

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Abstract

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The site-specific incorporation of transition metal complexes within DNA duplexes, followed by

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their immobilization on a gold surface, was studied by electrochemistry to characterize their

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ability to mediate charge. Cyclic voltammetry, square-wave voltammetry, and control

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experiments were carried out on fully matched and mismatched DNA strands that are mono- or

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bis-labeled with transition metal complexes. These experiments are all consistent with the ability

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of the metal centers to act as a redox probe that is well-coupled to the DNA π-stack, allowing

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DNA-mediated charge transport.

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Introduction

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The sequence-specific binding of DNA that provides the foundation of genetics has recently

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emerged as a powerful self-assembling principle. Two- and three-dimensional DNA structures

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and networks have been created using this strategy with exquisite control and complexity, and

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are being examined as potential tools in areas ranging from biophysics and medicine to materials

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science.1-5 In parallel, the DNA double helix has been shown to display charge transport over

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long distances under particular conditions.6 This property offers the possibility to ‘wire’ DNA

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nanostructures for applications in molecular electronics, artificial photosynthesis and biological

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sensing.7, 8 However, this has not been achieved in current DNA self-assembled structures, where

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DNA merely acts as a passive organizational template.

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A fundamental requirement to achieve this goal is to use redox probes that are intimately

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coupled electronically to the DNA π-stack. Transition metals would be ideal probes in this

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respect, because of their highly tunable electronic and photophysical properties as well as diverse

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geometries. Previous work by Barton and others has demonstrated the ability of intercalators and

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metallointercalators to efficiently access the DNA π-stack, resulting in DNA-mediated charge

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transport over distances as long as 34 nm.6,

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insertion of metal-DNA base pairs into break junctions and a significant increase in conductance

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as a result of metallation.10 However, neither of these systems have been used to build self-

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assembled 2D- or 3D-DNA nanostructures. Electrochemical data for non-intercalating metal-

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DNA conjugates has so far been restricted to ferrocene-DNA assemblies.11, 12 Rather than direct

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mediation of the DNA base pair stack, charge transport to ferrocene in these systems has been

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Shionoya, Guo et al. have recently shown the


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interpreted as arising from dynamics of the DNA strands and linkers that bring the redox probe

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in close proximity to the electrode for heterogeneous electron transfer.11, 12

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We have previously shown the site-specific incorporation of redox-active transition metals into

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DNA nanostructures.13 To achieve this, we created a structure where two ligands are in close

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contact with the DNA base stack, allowing the DNA double helix and the metal complex to

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synergistically stabilize each other (Scheme 1). This results in metallated DNA junctions of very

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high stability, chirality transfer from DNA to the metal environment, and the ability to build two-

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and three-dimensional structures, such as metal-nucleic acid cages and nanotubes.14,

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building strategy also allows introduction of different transition metals in specific DNA sites,

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using selective ligand environments for each metal.16 If these metal junctions were capable of

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accessing the DNA-base stack and acting as electrochemical ‘relays’, then this would allow the

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creation of metal-nucleic acid nanostructures with open charge conduction pathways at selected

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spots.

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This

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Herein we demonstrate through electrochemical measurements that these site-specifically

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incorporated transition metals are electronically coupled to the DNA base stack. For this, we use

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a modular approach to build DNA duplexes containing one or two CuI centres coordinated to

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2,9-bis-1,10-diphenyl phenanthroline (dpp) ligands. Interestingly, this allows the fine modulation

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of charge mediation without altering DNA structure. We find that an immobilized DNA duplex

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labeled with one copper center can mediate the charge involved in CuI to CuII oxidation. When

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labeled with two copper centers, the anodic current shows a two-fold increase, even though the

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distal copper is ca. 20 nm away from the electrode. Introducing one DNA base mismatch

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between the two copper centers decreases the anodic current to a value close to the mono-

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metallated complex, consistent with inhibited charge transport to the copper positioned further


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from the Au surface. Placing two mismatches in the DNA duplexes results in a significant (e.g.,

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82%) reduction of the current. To our knowledge, externally attached transition metals

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complexes (i.e. ferrocene) or intercalated luminescent (i.e. Ir(bpy)(phen)(phi)3+) metal-DNA

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conjugates have been the main focus when studying DNA charge mediation. This is the first

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report where DNA charge mediation is electrochemically demonstrated for a metal-DNA

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junction that can form nanostructures. This synergistic coupling can help assess DNA charge

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transport mechanisms and will lead to the assembly of complex nanostructures with opened

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charge conduction pathways.10

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Results and Discussion

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Scheme 1. Schematic representation of DNA-assemblies on gold; a) A1 ((O2, O3, O1)

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unmodified strands); b) A2 ((O4, O5, O1) CuI-(dpp)2 modified strands); A3 ((O4, O6, O1)

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CuI-(dpp)2 modified strands containing two CA mismatches); c) A4 ((O4, O7, O8, O1) CuI-

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(dpp)2 modified strands); A5 ((O4, O9, O8, O1) containing two CA mismatches); A6 ((O4,


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O10, O8, O1) containing one CA mismatch); A7 ((O4, O11, O8, O1) containing one CA

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mismatch).

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Our initial strategy involves the design of three different assemblies (Scheme 1, assemblies

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A1-A3). Each of the constructs contains a constant strand (O1) that acts as an anchor to the gold

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surface. O1 is an 18-mer modified at its 5’ terminus with a cyclic disulfide moiety.17 It contains a

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15-base binding region, as well as a short spacer consisting of three unhybridized thymines

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between the binding region and the disulfide, that ensures a small degree of conformational

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flexibility to the system when immobilized on the gold surface. In the first construct A1, strand

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O1 is bound to strands O2 and O3, which are unfunctionalized. In construct A2, O1 is bound to

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O4 and O5, each of which contain a single diphenyl phenanthroline (dpp) ligand. Hybridization

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of O4 and O5 orients the two dpp ligands into a tetrahedral pocket that is ideally suited to bind

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copper (I) atoms.13-16 Construct A3 is similar to A2, but features two CA mismatches along the

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DNA duplex. Although it has been shown that the addition of a single mismatch in the DNA

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sequence can give a two to three-fold decrease in charge mediation18 depending on its nature and

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position, in order to guarantee a noticeable signal difference for initial experiments, two

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mismatches were incorporated. The self-assembly of these units results, for all the structures, in a

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nick in the sugar-phosphate backbone. However, this has been previously shown not to interfere

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with charge transfer.19 Details of sequence design, synthesis of the oligonucleotides and

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incorporation of the phosphoramidite derivative of the dpp ligand,20 as well as assessment of

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oligonucleotide purity by denaturing polyacrylamide gel electrophoresis (PAGE), MALDI-TOF

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analysis, HPLC, circular dichroism and thermal denaturation studies can be found in the

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Supporting Information.


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The hybridization of constructs A1, A2, A3 as well as metallated derivatives A2Cu and A3Cu

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was carried out by annealing in phosphate buffer, and quantitative assembly yields were verified

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by non-denaturing PAGE. For metallated A2Cu and A3Cu, strands O4 with O5 and O4 with O6

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were first hybridized, respectively, then 1.1-1.3 equiv. of [Cu(CH3CN)4]PF6 were added. This

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was followed by addition of strand O1 at room temperature, in order to avoid binding of the CuI

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to its disulfide moiety. For these metallated assemblies, non-denaturing PAGE shows that the

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hybridization of the components into a single structure is preserved. When placed on a

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denaturing PAGE, A1, A2 and A3 disassembled into their components (Figure 1a; lanes 7, 8, 10)

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in the absence of metal (O1 and O4 have similar mobility and appear as a single band).

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Importantly, denaturing PAGE for the metallated assemblies A2Cu and A3Cu shows a lower

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mobility band that is assigned to the metal complex O4O5Cu (or O4O6Cu), which partly resists

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denaturation (Figure 1a; lanes 9, 11).13-16 Copper coordination was verified by circular dichroism

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(CD), which showed a positive peak at 345 nm, consistent with the formation of the CuI(dpp)2

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complex and transfer of chirality from the B-DNA duplex towards the copper complex, and by

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thermal denaturation analysis, which showed significant stabilization upon metallation (section

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S5.3).13-16


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Figure 1. a) Denaturing PAGE (15%, 4M urea) analysis of the oligonucleotide strands and of the

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assemblies with and without CuI; O2 (lane 1); O4 (lane 2); O1 (lane 3); O5 (lane 4); O6 (lane 5);

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O7 (lane 6); A1 (lane 7); A2, no CuI (lane 8); A2Cu (lane 9); A3, no CuI (lane 10); A3Cu (lane

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11). A1, A2 and A3 dissociate completely to their components, whereas the metallated A2Cu

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and A3Cu resist denaturation and show lower mobility bands (lanes 9, 11). b) Native PAGE

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(7%) analysis of the assemblies: A1 (lane 1); A2, with and without CuI, respectively (lanes 2, 3);

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A3 with and without CuI, respectively (lanes 4, 5). c) CD of O4O5 with (solid line) and without

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(dashed line) CuI.

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Prior to immobilisation on gold surfaces, solution-based electrochemical measurements were

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performed on the metallated DNA constructs and constituent units. We first studied a model

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complex of CuI(dpp)2PF6 by cyclic voltammetry in dry acetonitrile. We observed an E1/2 of 627

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mV vs SCE (corrected against (hydroxymethyl)ferrocene) for the CuI/CuII oxidation of the model

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complex (Figure S7), which corroborates comparable assignments in literature.20 We then


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assembled O4O5Cu and performed square-wave voltammetry due to its enhanced signal to noise

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ratio, in a 25 µL drop of PBS containing 15 µM solution of only the strands O4O5. The

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oligonucleotide O1 was disregarded in order to prevent disulfide adsorption on the working

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electrode (WE). DNA duplex integrity and presence of CuI in the dpp pocket for both matched

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and mismatched strands was verified by CD, thermal denaturation analysis and PAGE

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denaturing gels prior to and after solution electrochemistry to ascertain that no degradation

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occurred (see sections S5.3, S6.5 and Figure S15). Upon covalent modification of DNA with the

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CuI(dpp)2 complex, the oxidation and reduction waves are cathodically shifted by 195 mV

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resulting in E1/2 assignment of 432 mV (Figure 2a and S15). Although solvent dielectric constant

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changes can have an influence,21 this shift is also likely a result of the polyanionic DNA

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phosphate backbone acting as counteranions to facilitate CuI(dpp)2 oxidation.22 In addition,

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quasi-reversible behavior was observed when performing a reverse scan (Figure 2a, full line).

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Figure 2. a) Forward and reverse square-wave voltammograms of 25 µL of 15 µM solution of

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the O4-O5 assembly complexed with CuI (solid line) and self-assembled A2Cu on gold slides

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(dashed line) recorded at 10 mV/s in PBS (pulses height of 25 mV, pulses width of 50 ms and


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step height of 1 mV). For solution-based electrochemistry a Pt: WE, Pt:CE and Ag|AgCl as a

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reference electrode were used. b) Square-wave voltammogram of self-assembled DNA on gold

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slides in PBS. A2Cu (i), A3Cu (ii), A1 (iii), measured using the gold slide as the WE, Pt as the

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CE and Ag|AgCl as the reference electrode and background corrected from non-faradaic current

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contribution (see non-faradaic processes correction in Figure S14).

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Having identified a suitable potential window to monitor the CuI to CuII oxidation of the

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metallated DNA, the assemblies A1-A3 were investigated on a gold surface. A 10 µL aliquot of

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each DNA assembly solution (15 µM) was incubated on the gold surface for 2 days in a humidity

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box. Deposition efficiency (81 ± 5% based on the overall DNA added), calculated from the

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integrated charge of the disulfide reduction peak (-600 mV vs SCE) (as of example see Figure

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S10 for A5Cu2), was obtained following electrochemical reductive desorption of thiols

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performed in a 0.5 M solution of KOH.23 This experiment was carried out on immobilized DNA

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assemblies prior to incubation in a 1 mM mercaptohexanol diluent used to reduce background

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current and to favour the upright orientation of the assemblies during electrochemical

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measurements.

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As expected, for the assembly A1 where neither ligand nor CuI were present, no oxidation

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wave is detected (see Figure 2b-(iii)). In the case of the assembly A2Cu in the presence of the

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CuI(dpp)2 pocket, as illustrated in Figure 2b-(i), the oxidation wave potential is comparable to

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that observed in solution for the assembly O4O5Cu (Figure 2a). Interestingly, no reduction wave

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is observed for A2Cu in comparison to O4O5Cu (Figure 2a, dashed line). A similar

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voltammogram shape was obtained as the potential was cycled three times between -200 and 700

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mV (see Figure S8), thus confirming that the immobilized assemblies were stable in this

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experiment. This observation indicates that attachment to the gold surface does not interfere with


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the CuI electrostatically. By integrating the area under the curve at 430 mV, corresponding to

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assembly A2Cu in the voltammogram, it was found that 51 ± 4% (n = 3 at 95% confidence level)

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of CuI introduced in the oligonucleotides solution are being oxidized after deposition on the

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surface. Correcting for the deposition efficiency (see above), this corresponds to ~63% of the

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metals in the immobilized assemblies. These observations suggest that the DNA duplex may be

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able to mediate charge, and that the CuI(dpp)2 can access the DNA π-stack array.

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Charge transport was then investigated using assembly A3Cu, in which two C-A

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mismatches were included in order to partially disrupt the π-stack.6, 19 An important damping of

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82% in the integrated area under the curve at 430 mV, representing the CuI oxidation charge, is

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observed when these mismatches were included along the duplex (Figure 2b-(ii)).6, 11 A minor

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shift in electrochemical potential was measured between A2Cu and A3Cu and could be a result

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of a change in film resistance and/or DNA duplexes flexibility.11, 24, 25 We verified the stability of

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the mismatched DNA strands by carrying out thermal denaturation analysis of all complexes,

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demonstrating the observed damping was not due to strand dissociation (section S5.3).

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We also carried out a control experiment to verify that the homogeneity of monolayer

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deposition is unchanged between matched A2Cu and mismatched A3Cu assemblies. This is

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achieved using a redox mediator ([Ru(NH3)6]Cl3, RuHex)26 due to its known reversible

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electrochemical behaviour. Cyclic voltammograms were recorded at different scan rates for

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samples A2Cu and A3Cu in a 1.8 mM RuHex in PBS solution. The anodic peak current

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remained constant between the different samples (within a 1% error margin) (Figure S11)

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indicative of a similar deposition.26 Thus, A2Cu and A3Cu show similar deposition homogeneity

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and differences in their transport abilities are not due to different surface homogeneity. As in the

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case of A2Cu, the homogeneity of the deposited film on gold surface for the construct A5Cu2


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was evaluated using RuHex. The A5Cu2 voltammogram of the RuII/RuIII couple showed an

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anodic peak current similar to A2Cu (within a 10 % error margin), indicating similar film

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homogeneity for the longer DNA assemblies.

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The modular approach reported here can be readily adapted to site-specifically include

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additional CuI(dpp)2 pockets within the same structure, in order to tune the mediated charge

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transfer. In assemblies A4 to A7, two CuI(dpp)2 pockets have been incorporated (Scheme 1).

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Assembly A4Cu2 is a fully matched bis-metallated duplex. A6Cu2 is bis-metallated but contains

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a mismatch between the metals. A7Cu2 has a mismatch between the electrode and the proximal

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copper (closest to the electrode) and finally, A5Cu2 has two mismatches, one before and one

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after the proximal copper (see supporting information for sequences). All DNA duplex segments

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preceding and following the proximal CuI(dpp)2 contain 30 base pairs.

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The cyclic voltammograms for assemblies A2Cu (i), A4Cu2 (ii), A5Cu2 (iii), A6Cu2 (iv) and

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A7Cu2 (v) are shown in Figure 3. For the mono-metallated A2Cu, a peak assigned to the CuI/CuII

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oxidation of the CuI(dpp)2 pocket is observed at 446 mV, consistent with the potential range in

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Figure 2a-b. The observed CuI/CuII oxidation of fully matched, bis-metallated A4Cu2 is shifted

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anodically and is observed at 500 mV. This is in agreement with existing literature on the effect

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of vicinal electrochemical centres in tethered molecules assemblies.27 According to the Robin-

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Day classification of mixed-valence compounds, A4Cu2 would either belong to class II or III as a

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single oxidation wave is recorded. This is associated with an intrinsic charge delocalization and

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electronic communication between each of the metal atoms. This observation still remains to be

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verified by changing the inorganic moieties in the assemblies and performing a spectroscopic

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analysis.


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Interestingly, following background correction, the integrated charge ratio of A4Cu2 to A2Cu

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was 2.18, corresponding to oxidation of two CuI(dpp)2 pockets incorporated within the DNA

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helix. When a mismatch is introduced between the two copper centers, the anodic current is

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found to decrease to a value close to the A2Cu. This is consistent with inhibited charge transport

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to the second copper center (iv, integrated charge ratio A4Cu2:A6Cu2 1.84:1). The introduction

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of a mismatch between the electrode and the proximal CuI(dpp)2 impedes the CuI/CuII oxidation

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even further (77% of A4Cu2). Finally, when two mismatches are placed before and after the first

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copper center (A5Cu2, iii), no oxidation wave is detected for the copper centers, consistent with

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hindered charge transport.

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Figure 3. Cyclic voltammograms in PBS of self-assembled DNA on gold slides. A2Cu (i)),

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A4Cu2 (ii)), A5Cu2 (iii)), A6Cu2 (iv)) and A7Cu2 (v)) background corrected from non-faradaic

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current contributions. The gold slide was used as a WE, Pt as the CE and Ag|AgCl as the

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reference electrode. The voltammograms were recorded at 50 mV/s.


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The results presented in Figure 3 are consistent with DNA-mediated charge transport to the

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copper centres that is relatively distance-insensitive, as well as efficient coupling of the DNA

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base stack to both metals – the distal metal is in fact 63 DNA bases or ~ 20 nm away from the

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electrode.

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We had previously observed that when DNA duplexes possessing (dpp)2 pockets (such as in

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assembly A2) are titrated with CuII rather than CuI, spontaneous reduction of the metal to CuI

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occurs.15 This was clearly detected by CD in phosphate buffer and in the absence of a reducing

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agent. The source of the electron in this process was postulated to be one of the bases in the

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adjacent DNA strand, suggesting DNA-mediated charge transport.15 Our present electrochemical

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experiments on gold surfaces are consistent with this hypothesis. While oxidation and reduction

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waves for unbound O4O5Cu are observed in Figure 2a (solid line), in the gold immobilized

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assembly A2Cu no reduction wave of the oxidized CuII could be observed (Figure 2a, dashed

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line). While more data is required to examine this mechanism (i.e. sequential treatment in hot

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piperidine followed by denaturing gel electrophoresis28), it is possible that oxidation at one of the

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adjacent bases of the DNA duplex A2Cu (e.g., guanines) causes rapid intramolecular CuII

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reduction back to CuI before potential switching.

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The above observations, namely i. anodic current from A2Cu and A4Cu2 shows a two-fold

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increase in presence of two CuI(dpp)2 pockets, ii. decrease in the current to values close to mono-

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metallic complexes with a mismatch between the two metals (A4Cu2 vs A6Cu2), and iii. absence

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of oxidation when two mismatches are introduced (A4Cu2 vs A5Cu2) are thus consistent with

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DNA-mediated charge transport to the CuI(dpp)2 pocket within these metal-DNA assemblies.[i]

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_____________________________________________________________________________________________________________________

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[i] : While we cannot fully exclude that the positive potential scan allows the DNA strands to approach the gold surface, our results give strong evidence of charge mediation by the DNA duplex, rather than direct oxidation of the copper by the electrode. To evaluate the efficiency of charge transfer, Laviron’s analysis requires a reversible


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anodic/cathodic wave separation of 200 mV,29 even at high scan rates (> 250 mV/s), where the system should be entering the totally irreversible reaction regime. However, the CuI(dpp)2 oxidation/reduction peak was found to be irreversible.

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Conclusion

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In conclusion, we have reported a modular approach to construct metal-DNA hybrid

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assemblies without altering the DNA helical structure, where the metal-ligand environments can

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probe and interact with the π-stack array, thus allowing for charge mediation. The different

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building blocks assembled here allow fine tuning of the current transported, by varying the

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number of CuI(dpp)2 pockets in the DNA duplex and the position of DNA mismatches. The

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metal-dpp pockets can be incorporated at any position and in any number into DNA strands, and

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can be used to access two-and three-dimensional DNA nanostructures.13-16 We anticipate this

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methodology to readily provide metal-DNA probes that modulate and possibly ensure direct

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charge transport across DNA nanostructures rather than a collisional mechanism where the metal

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complex would be directly in contact or in close proximity to the electrode surface.

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ASSOCIATED CONTENT

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Supporting Information.

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Details about synthesis and DNA assembly as well as electrochemical characterization and

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experimental conditions can be found in Supporting Information. This material is available free

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of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

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Corresponding Author

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* E-mail: [email protected] or [email protected]


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Author Contributions

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‡These authors contributed equally.

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ACKNOWLEDGMENT

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We thank the NSERC, CFI, CSACS and CIFAR for financial support.

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REFERENCES

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Table of Contents Entry

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A modular approach is used to assemble double stranded DNA modified with bisphenanthroline

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ligands complexed to CuI. The complex is site-specifically inserted within the π-stack to enable

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charge conduction when grafted on gold surfaces. Electron mediation is monitored using

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electrochemistry and can be finely modulated through the addition of mismatches and an extra

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CuI center. This approach is promising for future nanomaterial applications.

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Modulation of Charge Transport Across Double-Stranded DNA by the

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