Electronic Supporting Information

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1 1S Electronic Supporting Information DNA-mediated Electron Transfer in DNA Duplexes Tethered to Gold Electrodes via Phosphorothioated da Tags Rui Campos,, Alexander Kotlyar and Elena E. Ferapontova *,, Interdisciplinary Nanoscience Center (inano) and Center for DNA Nanotechnology (CDNA) at inano, Science and Technology, Aarhus University, Gustav Wieds Vej 14, 8000, Denmark Department of Biochemistry and Molecular Biology, George S. Wise Faculty of Life Sciences and The Center of Nanoscience and Nanotechnology, Tel Aviv University, Ramat Aviv 69978, Israel Additional experimental details Materials. DNA sequences synthesized by Metabion Int., Martinsried, Germany were as follows (from 5 to 3 ): probe DNA with a 3 C6-disulfide (HO-(CH 2 ) 6 -S-S-(CH 2 ) 6 -) modification (C6- DNA): GTT GTG CAG CGC CTC ACA AC C6 S-S-C 6 OH; probe DNA with da 5 * tag (da 5 *- DNA): GTT GTG CAG CGC CTC ACA AC A*A*A*A*A*; complementary DNA (cdna): GTT GTG AGG CGC TGC ACA AC; and SNP-containing cdna: GTT GTG AGG CAC TGC ACA AC. Components of buffer solutions, tris(2-carboxyethyl)phosphine hydrochloride (TCEP), methylene blue (MB) and hexaammineruthenium (III) chloride ([Ru(NH 3 ] 6 Cl 3 ) were purchased 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 electrodes (CH Instruments, Austin, Texas, USA; diameter 2 mm) were 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 ethanol:water solution for 10 minutes. Then the electrodes were

2 2S electrochemically polished in 1 M H 2 SO 4 and 1 M H 2 SO 4 /10 mm KCl. The electrochemical surface area was determined from the gold surface oxide reduction peaks in 0.1 M H 2 SO 4. Before DNA immobilization, the electrodes were kept in absolute ethanol for 30 minutes. DNA was immobilization was performed by overnight incubation of the electrodes in a 10 μm dsdna solution (1 h hybridization with 15 μm cdna in 10 mm phosphate buffer solution, PBS (sodium salt), ph 7.0, containing 50 mm NaCl and 100 mm MgCl 2 ) while reacting with TCEP for disulphide reduction (da 5 *-linked DNA was not treated with TCEP). The dsdna-modified electrodes were thoroughly rinsed with 10 mm PBS, ph 7.0, incubated in the 2 mm MCH/10 mm PBS solution for 30 min, and then either immediately used in electrochemical experiments or kept at 4 C in 10 mm PBS, ph 7. The DNA surface coverage is referred to the electrochemically active electrode surface area of 55±05 cm 2. In studies of the effect of DNA SNP on ET rates, the dsdna-modified electrodes (dsdna composed of fully complementary sequences) were dehybridized by thorough washing with water and after that overnight re-hybridized with the SNPcontaining cdna strand (10 μm solution). Instrumentation and Procedure. Cyclic voltammetry and square wave voltammetry were performed in a three-electrode cell made of a dark glass connected to the potentiostat μautolab type III (Eco Chemie B. V., Utrecht, Netherlands) equipped with GPES 4.9 and with NOVA 1.8 software. An Ag/AgCl (3.5 M KCl) electrode was the reference electrode and a platinum flag (1 cm 2 ) was the auxiliary electrode. The 10 mm PBS working solution, ph 7, contained 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 determined with 0.2 mm Ru(NH 3 ) 3+ 6.

3 3S Figures Electrode modification i / µa -0.2 i / µa Figure S1. Representative CVs of the da* 5 -DNA-modified gold electrode, (left) before and (right) after blocking the electrode surface with mercaptohexanol. Potential scan rate is 750 mv s -1 and the DNA surface coverage is 1.3 pmol cm -2.The mercaptohexanol treatment removes nonspecifically adsorbed DNA (surface-bound not via the linker) that is nevertheless also capable of interactions with methylene blue and may produce electrochemical signal not related to the DNA-mediated ET.

4 4S DNA surface coverage measurements Figure S2. Representative chronocoulometric data for (1) C6-tethered DNA, fully complementary duplex; (1 ) C6-tethered SNP-containing DNA duplex; (2) da 5 *-tethered DNA, fully complementary duplex;, (2 ) da 5 *-tethered SNP-containing DNA duplex; (1(4)) C6-tethered DNA, fully complementary duplex after 4 days of storage and (2(4)) da 5 *-tethered DNA, fully complementary duplex after 4 days of storage, in the absence (back stands for background) and presence of 200 μm Ru(NH 3 ) 6 3+.

5 5S Figure S3. Representative background-corrected CVs, recorded with the DNA-modified gold electrodes in 10 mm PBS, ph 7, containing 0.1 μm MB, the current is normalized for the surface coverage Γ (pmol cm -2 ), scan rate v (V s -1 ) and electrode area A (cm 2 ), for (A) the C6 and (B) da5* linkers. (C, D) Dependence of the normalized relation (i/f) between the SWV peak currents i and the frequency f in semi-logarithmic coordinates, (C): the C6 linker (full symbols and lines) and (D): the da5* linker (empty symbols and dashed lines), all other conditions are the same as in (A).

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7 7S Representative Cyclic and Square Wave Voltammograms Figure S4. Representative CVs for (A) C6-tethered DNA, fully complementary duplex; (B) C6- tethered SNP-containing DNA duplex; (C) da 5 *-tethered DNA, fully complementary duplex; and (D) da 5 *-tethered SNP-containing DNA duplex The insets show the linear dependence of the current with the scan rate.

8 8S A 2.5 B I / µa 1.0 -I / µa f / Hz 400 f / Hz C 1.0 -I / µa D 0.6 -I / µa f / Hz 400 f / Hz Figure S5. Representative SWVs for (A) C6-tethered DNA, fully complementary duplex; (B) C6- tethered SNP-containing DNA duplex; (C) da 5 *-tethered DNA, fully complementary duplex; and (D) da 5 *-tethered SNP-containing DNA duplex.

9 9S Analysis of ET rate constants from CV: ET rate constants k s were evaluated by the Laviron method, from the peak potentials E peak values or, more precisely, from the overpotential/(cathodicanodic) peak potential separation. 1 According to Laviron there are two possibilities to calculate the k s of the quasi-reversible ET in the case of adsorbed redox species. 2 For peak separations >200/n mv, the k s is determined by extrapolation of the linear part of the anodic and cathodic branches of the E peak - log ν dependence: k s =αnfν c /RT=(1-α)nFν a /RT (1) where α is the transfer coefficient and ν c and ν a are the intercept points of the extrapolated lines at ΔE=0. α is calculated from the slope of the linear part of the E peak - log ν dependence slope =-2.3RT/(αnF) ; slope =2.3RT/((1-α)nF) (2) or, alternatively, from log ν a log ν c =log (α/(1-α)) (3) For peak separations <200/n mv the k s can be estimated by the following equation m=(rt/f)(k s /(nν)) (4) where m values are tabulated. 2 Analysis of ET rate constants from SWV: Analysis of k s was also performed by processing the SWV data within the Komorsky-Lovrić Lovrić formalism. 3 SWVs corresponding to surfaceconfined process are highly sensitive to kinetics of ET 3-4, and its rate may be determined by: k s =ω max f max (5) where f max is the critical frequency, at which the relation of the measured current I to the frequency at which it is measured f (I/f) is maximal, and ω max is the kinetic parameter that depends both on α and ne sw 3-4. References (1) Bard, A. J.; Faulkner, L. R. Electrochemical methods: fundamentals and applications; 2nd ed.; John Wiley & Sons: New York, (2) Laviron, E. J. Electroanal. Chem. Interfac. 1979, 101, 19. (3) Lovrić, M.; Komorsky-Lovric, Š. Journal of Electroanalytical Chemistry and Interfacial Electrochemistry 1988, 248, 239. (4) Mirceski, V.; Komorsky-Lovric, S.; Lovric, M. Square-Wave Voltammetry: Theory and Application; Springer, 2007.