Copyright 2008 American Chemical Society
MAY 6, 2008 VOLUME 24, NUMBER 11
Letters Interaction of Viologens with Nucleic Acid G-Tetrades Simona Asaftei,* Helmut Rosemeyer, and Lorenz Walder Institute of Chemistry, UniVersity of Osnabru¨ck, Barbarastrasse 7, 49076 Osnabru¨ck, Germany ReceiVed December 21, 2007. In Final Form: April 7, 2008 The interaction between the tetrade-forming oligonucleotide 5′-d(T4G4T4) and monoalkylated bipyridinium salts, such as 1-ethyl-4-pyridin-4-ylpyridinium bromide, is reported. The oligomer forms tetrades in the presence of K+ ions but not with Li+. Additionally, the interaction of the thrombin-binding aptamer 5′-d(GGTTGGTGTGGTTGG) (TBA) with a dialkylated bipyridinium salt, viologen, was studied by cyclic voltammetry. This was performed either on a TiO2 electrode, derivatized with 3-aminopropyltriethoxysilane (APS), using [Fe(CN)6]4- as a marker ion or without a marker ion on an electrostatically TiO2-bound amino-ferrocenyl derivative. Both experiments proof a strong interaction between the immobilized aptamer and the viologen. Third, the electrochemical response of the specific thrombin binding to the immobilized aptamer was studied.
Introduction G-Tetrades are important for the regulation of biochemical processes in the telomere region of the chromosome. During the past decade, a series of N-heteroaromatic compounds were identified to bind to and to stabilize G-quadruplexes either by stacking interaction or by being laid out onto the nucleic acid aggregate. It had been shown that the oligomer 5′-d(T4G4T4) forms tetrades in the presence of Na+ and particularly K+, but not in the presence of Li+.1 In preliminary experiments we found that this oligomer forms also tetrades in the presence of a monoalkylated bipyridinium salt, namely, 1-ethyl-4-pyridin-4-ylpyridinium bromide. Ionexchange high-performance liquid chromatography (HPLC) exhibits two peaks, of which the one with the slower mobility represents the quadruplex aggregate form of the oligomer. The latter as well as its relative stabilities in the presence of alkali * Corresponding author. Address: Institute of Chemistry, University of Osnabru¨ck, Barbarastr. 7, D-49076 Osnabru¨ck, Germany. E-mail: sasaftei@ uos.de. Phone: *49 (0) 541 969 2389. Fax: *49 (0) 541 969 2370. (1) Davis, J. T. Angew. Chem. 2004, 116, 684–716.
ions had been proved by intensive HPLC studies and by temperature dependent CD measurements.2,3 The results prompted us to test the interaction of the dialkylated bipyridinium salt (viologens), namely 1,1′-bis(2ammoniopropyl)-4,4′-bipyridinium tetrabromide (DAPV) and the oligonucleotide 5′-d(GGTTGGTGTGGTTGG), a thrombinbinding aptamer [TBA], for their G-quadruplex forming properties on electrodes and as an electrochemical sensing principle. Artificial ion channel sensors have been successfully applied to DNA hybridization. They are based on the modulation of the current by electrostatic interactions of surface channels and highlycharged electroactive species in solution such as Fe(CN)63-.4 Small changes in the surface charge of the channels–even those restricted to the orifices of the channels in mesoporous TiO2–can have a large influence on the observed Fe(CN)63- current. (2) Neidle, S.; Parkinson, G. Nat. ReV., Drug DiscoVery 2002, 1, 383–393. (3) Seela, F.; Wei, C. F.; Melenewski, A.; Feiling, E. Nucleosides Nucleotides 1998, 17(9-11), 2045–2052. (4) Scho¨n, P.; Degefa, H. T.; Asaftei, S.; Meyer, W.; Walder, L. J. Am. Chem. Soc. 2005, 127(32), 11486–11496.
10.1021/la704001n CCC: $40.75 2008 American Chemical Society Published on Web 05/06/2008
5642 Langmuir, Vol. 24, No. 11, 2008
Letters
Figure 2. Cyclic voltammogram of the thrombin-binding aptamermodified TiO2 electrodes (a) TBA-K+ (dashed line) and (b) TBADAPV4+ (solid line) in 1 mM K4[Fe(CN)6]/1 M NaClO4, vs Ag/AgCl, at V ) 20 mV s-1.
Figure 1. Preparation of a TiO2 electrode coated with primary amino groups.
Material and Methods The TiO2 electrodes were prepared by the doctor blade method on fluorine-doped tin oxide (FTO) glass slides.5 The TiO2 nanocrystals (20 nm size) built up a mesoporous film (ca. 5 µm thick), which was functionalized with 3-aminopropyltriethoxysilane (APS), according to the method reported by Lee et al. (Figure 1).6 The silanization of the TiO2 surface was checked electrochemically after reaction of the amine-coated surface with ferrocenyl carboxylic acid fluoride. From the CV response, the surface concentration of the ferrocenyl moieties was found to be 3.6 × 10-9 mol/cm2.
Results and Discussion In the following, the APS-TiO2 electrodes were exposed to solutions of the thrombin binding aptamer in the presence of different cations, i.e., TBA-K+, TBA-Li+, TBA-DAPV4+, for 1 h at room temperature. Notably, electrostatic binding of the negatively charged aptamer on the positively charged APSTiO2 surface was envisaged. The electrochemical experiments were done either in the presence of a millimolar solution of [Fe(CN)6]3-/4- or in pure electrolyte. The marker ions [Fe(CN)6]3-/4- undergo an electron transfer only at the FTO layer, i.e., they have to diffuse through the mesoporous system or they have to shuttle electrons via lateral electron hopping through the channels. We expected a substantial modulation of this marker ion current as a result of the electrostatic interaction of Fe(CN)6]3-/4- with the aptamer sitting on channel walls. In the first experiment, the marker ion current responses of TBA-DAPV4+ and TBA-K+ were compared (Figure 2). The current response of the TBA-DAPV4+ is larger than the current response of TBA-K+, and a typical viologen signature at -0.45 V is observed. The presence of viologen is strong evidence of a stable TBA-DAPV4+ complex. The peak at -0.45 V represents reduction of DAPV4+ through the TiO2 semiconductor. An oxidation via the same pathway is not possible; it occurs by electrocatalysis through FeIII/II(CN)6 in accordance with the larger anodic current in case b at ca. 0.30 V. Further evidence for the stability of the TBA-DAPV4+ complex is presented in Figure 3. In this experiment without marker ions, the electrodes were investigated in pure electrolyte. The electrodes (5) Campus, F.; Bonhote, P.; Gra¨zel, M.; Heinen, S.; Walder, S. Sol. Energy Mater. Sol. Cells 1999, 56(3-4), 281–297. (6) Lee, G. S.; Lee, Y. J.; Ha, K.; Yoon, K. B. Tetrahedron 2000, 56(36), 6965-6968.
Figure 3. Cyclic voltammograms of thrombin-binding aptamer-modified TiO2 electrodes after exposition to AFDCA: (a) TBA-K+-AFDCA, (b) TBA-Li+-AFCDA, and TBA-DAPV4+-AFCDA. Inset: the same electrode with a blown-up current range. The typical ferrocene derivative current peak at ca. +0.3 V is not present. The electrodes were measured in 1 M NaClO4 aq. vs Ag/AgCl, at V ) 20 mV s-1.
were prepared as those discussed in the first experiment (Figure 2). After exposition to the thrombin-binding aptamer complex, the electrodes were additionally incubated in a solution of the ferrocene derivative 1,1′-bis[bis(aminoethyl)amino]ferrocene dicarboxamide (AFDCA · 6H+) for 1 hour at room temperature. This redox system is supposed to bind electrostatically to the aptamer surface. Remarkably, in the case of TBA-K+-AFDCA (a), a small ferrocene response is observed. It is consistent with the aptamers reaching far into the porous system and allowing electron transfer at the FTO electrode. The current response is smaller for TBA-Li+-AFDCA (b) and has disappeared for TBA-DAPV4+-AFDCA. The higher surface concentration of the ferrocene compound in case a, as compared to case b (Figure 3), could be related to a conformational change, i.e., possibly to the transition of the aptamer structure to the random coil. The lack of electroactivity at ca. +0.35 V in case c could by related to viologens sitting on the surface of the oligonucleotide and inhibiting ferrocene coordination. This is supported by the peak at -0.45 V, which is typical for viologen reduction. In Figure 4 we present the [Fe(CN)6]3-/4- marker ion response of the APS-TiO2-, TBA-K+-, and TBA-DAPV4+-modified
Letters
Langmuir, Vol. 24, No. 11, 2008 5643
onto APS-TiO2 could be an explanation for the current increase in case a. We cannot definitely explain why nonspecific adsorption (case a) and specific adsorption (case c) have an opposite influence on the current response. This could be related to different areas and surface charges in a and c of the thrombin exposed to the [Fe(CN)6]3-/4- flow.9
Conclusion
Figure 4. Cyclic voltammogram of thrombin binding aptamer modified TiO2 electrodes after exposition to a thrombin solution: (a) TiO2APS-thrombin (black line), (b) TBA-DAPV4+-thrombin (red line), (c) TBA-K+-thrombin (blue line), in 1 mM K4[Fe(CN)6]/1 M NaClO4 vs Ag/AgCl, at V ) 20 mV s-1.
electrodes, which were further incubated for 1 h in a solution of thrombin. In contrast to TBA, thrombin cannot enter the porous system of TiO2. Any complex formation between thrombin and the aptamer or a nonspecific adsorption of thrombin onto the electrode may influence the electrostatics at the channel orifices and modulate the marker ion current. A diminished current is observed in case c, as expected for the specific TBA-K+ thrombin interaction.7,8 In case b, with the TBA-DAPV4+ complex, the marker ion current is large and does not decrease after thrombin exposition of the electrode. Probably no TBA-DAPV4+ complex formation takes place. A nonspecific absorption of thrombin (7) Radi, A. E.; O’Sullivan, C. K. Chem. Commun. 2006, (32), 3432–3434. (8) Radi, A. E.; Sanchez, J. L. A.; Baldrich, E.; O’Sullivan, C. K. J. Am. Chem. Soc. 2006, 128(1), 117–124.
Mono- and dialkylated bipyridinium salts show complex formation with guanine containing nucleosides, similar to the well-known K+-, Na+-, and Cs+-promoted G-tetrade complexes. Complex formation can be followed by HPLC or, as shown for the first time, electrochemically. The aptamer is complexed with K+ or Vio+2 and then grafted on the surface of a TiO2 electrode. This device shows a current response toward thrombin. Similarly, molecular architectures on TiO2 surfaces may be used as electrochemical sensors based on modulated marker ion current as a consequence of analyte-membrane interaction.4,10 Acknowledgment. The work was financially supported by the research pool of the University of Osnabru¨ck. Supporting Information Available: Solid-phase oligonucleotide synthesis. Stock solution of TBA and TBA–K+, TBA–Li+, and TBA-1,1′-bis(2-ammonium-propyl)-4,4′-bipyridinium tetrabromide (DAPV)4+. Electrode surface modification. Analytical electrochemistry. Determination of surface concentrations. This material is available free of charge via the Internet at http://pubs.acs.org. LA704001N (9) Bang, G. S.; Cho, S.; Kim, B. G. Biosens. Bioelectron. 2005, 21(6), 863– 870. (10) Sugawara, M.; Kojima, K.; Sazawa, H.; Umezawa, Y. Anal. Chem. 1987, 59(24), 2842–2846.
