Langmuir 2009, 25, 3839-3844
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Characterization of Mismatched DNA Hybridization via a Redox-Active Diviologen Bound in the PNA-DNA Minor Groove Eli G. Hvastkovs*,† and Daniel A. Buttry‡ Department of Chemistry, East Carolina UniVersity, 300 Science and Technology Bldg., GreenVille, North Carolina 27858, and Department of Chemistry & Biochemistry, Arizona State UniVersity, PO Box 871604, Tempe, Arizona 85287-1604 ReceiVed October 6, 2008. ReVised Manuscript ReceiVed December 17, 2008 Diviologen molecules of the general formula CH3(CH2)11V2+(CH2)6V2+(CH2)11CH3 (C12VC6VC12, V2+ ) 4,4′bipyridinium or viologen) were employed to electrochemically assay DNA hybridization to PNA probes immobilized at Au electrodes. Immobilized 15-mer PNA probes were exposed to 25-mer DNA oligonucleotides containing either complementary or single base mismatched sequences. In the presence of complementary PNA-DNA hybrids, the V2+/+ redox couple of C12VC6VC12 exhibited a unique double-wave cyclic voltammogram, with a formal potential shifted -100 mV from the Ef in the presence of single base mismatched DNA hybrids or PNA probes alone. Integration of the CVs demonstrated that C12VC6VC12 exhibited binding cooperativity to the complementary PNA-DNA hybrids and saturated at a ratio of 2:1 (C12VC6VC12:hybrid). Reduced C12VC6VC12 (V+) absorption spectra showed a significant λmax blue shift (22 nm) in the presence of complementary hybrids compared to the λmax in the presence of PNA or mismatched DNA hybrids. Chronocoulometry was employed to assay surface populations and obtain thermodynamics for C12VC6VC12 binding. These data are consistent with C12VC6VC12 bound in the minor groove of complementary hybrids as face-to-face π-dimers. This approach to distinguishing complementary hybrids from mismatched hybrids is novel, with potential applications involving detection of DNA damage or single nucleotide polymorphism (SNP) analysis.
Introduction The analysis of short DNA oligomers of known sequences is important in the areas of genetics and medicine to elucidate the causes of various diseases. Damage to DNA at individual “hotspot” codons can lead to mutations that can potentially alter vital transcription products in fundamental manners1,2 while single nucleotide polymorphisms (SNPs) can be the direct cause of disease.3,4 Detection and identification of DNA sequences of interest are typically done via direct sequencing following enzymatic and amplification steps or via DNA hybridization biosensor strategies.5,6 Biosensor techniques are more convenient and typically involve surface immobilization of short known sequence DNA strands followed by target capture and transduction through an optical,7-10 mass,11,12 or electrochemical signal.4,13 * To whom correspondence should be addressed. E-mail: hvastkovse@ ecu.edu. † East Carolina University. ‡ Arizona State University. (1) Denissenko, M. F.; Pao, A.; Pfeifer, G. P.; Tang, M.-S. Oncogene 1998, 16, 1241–1247. (2) Denissenko, M. F.; Pao, A.; Tang, M.-s.; Pfeifer, G. P. Science (Washington, D.C.) 1996, 274, 430–432. (3) Hu, W.; Feng, Z.; Ma, L.; Wagner, J.; Rice, J. J.; Stolovitzky, G.; Levine Arnold, J. Cancer Res. 2007, 67, 2757–65. (4) Millan, K. M.; Mikkelsen, S. R. Anal. Chem. 1993, 65, 2317–2323. (5) Wang, J. Nucleic Acids Res. 2000, 28, 3011–3016. (6) Palecek, E.; Jelen, F. Crit. ReV. Anal. Chem. 2002, 32, 261–270. (7) Jordan, C. E.; Frutos, A. G.; Thiel, A. J.; Corn, R. M. Anal. Chem. 1997, 69, 4939–4937. (8) Nelson, B. P.; Grimsrud, T. E.; Liles, M. R.; Goodman, R. M.; Corn, R. M. Anal. Chem. 2001, 73, 1–7. (9) Brockman, J. M.; Frutos, A. G.; Corn, R. M. J. Am. Chem. Soc. 1999, 121, 8044–8051. (10) Ferguson, J. A.; Boles, T. C.; Adams, C. P.; Walt, D. R. Nat. Biotechnol. 1996, 14, 1681–1684. (11) Okahata, Y.; Matsunobu, Y.; Ijiro, K.; Mukae, M.; Murakami, A.; Makino, K. J. Am. Chem. Soc. 1992, 114, 8299–300. (12) Caruso, F.; Rodda, E.; Furlong, D. N.; Niikura, K.; Okahata, Y. Anal. Chem. 1997, 69, 2043–2049. (13) Drummond, T. G.; Hill, M. G.; Barton, J. K. Nat. Biotechnol. 2003, 21, 1192–1199.
Electrochemical detection platforms offer promise due to detection speed, miniaturization possibilities, label-free detection schemes, and instrumentation cost.13 Electrochemical DNA hybridization platforms can employ direct nucleobase oxidation via transition metal catalysts14-18 or indirect detection through the interrogation of molecules that bind to DNA.19-25 Many electrochemically active molecules that bind to DNA are cationic and bind electrostatically to negatively charged phosphate groups of the DNA backbone. Thermodynamically, an isolated single base mismatch on a 30-mer DNA hybrid does not greatly affect the stability of a DNA duplex. In addition, the effects of the mismatch are sequence and location dependent.26 Therefore, cationic small molecule redox reporters can potentially interact similarly with ssDNA, stable mismatched hybrids, or complementary hybrids making differentiation difficult. Intercalators that insert a planar moiety between adjacent base pairs of DNA may be used and can provide impressive selectivity and sensitivity.27-32 However, hydrophobic and electrostatic interactions may also cause these (14) Armistead, P. M.; Thorp, H. H. Anal. Chem. 2000, 72, 3764–3770. (15) Ontko, A. C.; Armistead, P. M.; Kircus, S. R.; Thorp, H. H. Inorg. Chem. 1999, 38, 1842–1846. (16) Napier, M. E.; Thorp, H. H. Langmuir 1997, 13, 6342–6344. (17) Johnston, D. H.; Thorp, H. H. J. Phys. Chem. 1996, 100, 13837–13843. (18) Johnston, D. H.; Glasgow, K. C.; Thorp, H. H. J. Am. Chem. Soc. 1995, 117, 8933–8. (19) Herne, T. M.; Tarlov, M. J. J. Am. Chem. Soc. 1997, 119, 8916–8920. (20) Steel, A. B.; Herne, T. M.; Tarlov, M. J. Bioconjugate Chem. 1999, 10, 419–423. (21) Steel, A. B.; Herne, T. M.; Tarlov, M. J. Anal. Chem. 1998, 70, 4670– 4677. (22) Hvastkovs, E. G.; Buttry, D. A. Langmuir 2006, 22, 10821–10829. (23) Pang, D.-W.; Abruna, H. Anal. Chem. 1998, 70, 3162–3169. (24) Pang, D.-W.; Abruna, H. Anal. Chem. 2000, 72, 4700–4706. (25) Kelley, S. O.; Barton, J. K.; Jackson, N. M.; Hill, M. G. Bioconjugate Chem. 1997, 8, 31–37. (26) Allawi, H. T.; SantaLucia, J. Biochemistry 1997, 36, 10581–10594. (27) Takenaka, S.; Uto, Y.; Saita, H.; Yokoyama, M.; Kondo, H.; Wilson, W. D. Chem. Commun. 1998, 1111-1112. (28) Takagi, M. Pure Appl. Chem. 2001, 73, 1573–1577. (29) Onfelt, B.; Lincoln, P.; Norden, B. J. Am. Chem. Soc. 1999, 121, 10846– 10847.
10.1021/la803284r CCC: $40.75 2009 American Chemical Society Published on Web 03/10/2009
3840 Langmuir, Vol. 25, No. 6, 2009 Scheme 1. Structure of C12VC6VC12
types of molecules to exhibit affinity for ssDNA probes as well.33,34 Overall, there is a continual interest for new and interesting approaches in the selective assay of DNA hybridization.32 In order to circumvent some of the nonspecific binding issues of cationic molecules for ssDNA, immobilized peptide nucleic acid (PNA) probes have been employed.35-41 PNA is a neutral DNA analogue consisting of a (2-aminoethyl)glycine backbone that is resistant to enzymatic degradation and forms stable helices with cDNA targets.42-44 PNA-DNA hybrids show enhanced thermodynamic stability compared to analogous dsDNA hybrids,43 and PNA exhibits increased stringency toward mismatch DNA target capture.45 Cationic molecules show diminished attraction toward the neutral probe surface, and enhanced electrochemical signals denote hybridization with anionic DNA. Immobilized PNA probes have allowed discrimination between proper hybrids and mismatch sequences employing cobalt35,40,41,46 or ruthenium compounds47 that exhibit electrostatic attraction toward DNA. Also, in situ PNA-DNA hybridization monitoring via the increased charge transfer resistance of Fe(CN)64- has been described.48 One key drawback to PNA is that traditional minor groove binders and intercalators designed to enhance the detection of DNA hybridization bind weakly if at all to PNA-DNA hybrids due to slight differences in helix structure.49 Thus, there is a need for redox reporters with improved binding capabilities to exploit the benefits of PNA for the use of DNA target capture. We recently described a DNA hybridization assay employing the redox active molecule C12VC6VC12 (Scheme 1) that was shown to give a unique electrochemical signal when bound in the dsDNA minor groove.22 The binding caused face-to-face π-dimer formation of cation radical viologen moieties in the minor (30) Wakelin, L. P. G.; Bu, X.; Eleftheriou, A.; Parmar, A.; Hayek, C.; Stewart, B. W. J. Med. Chem. 2003, 46, 5790–5802. (31) Hvastkovs, E. G.; Buttry, D. A., Anal. Chem. 2007, in press. (32) Garcia, T.; Revenga-Parra, M.; Abruna, H. D.; Pariente, F.; Lorenzo, E. Anal. Chem. 2008, 80, 77–84. (33) Rye, H. S.; Glazer, A. N. Nucleic Acids Res. 1995, 23, 1215–1222. (34) Hvastkovs, E. G.; Buttry, D. A. Anal. Chem. 2007, 79, 6922–6926. (35) Wang, J.; Palecek, E.; Nielsen, P. E.; Rivas, G.; Cai, X.; Shiraishi, H.; Dontha, N.; Luo, D.; Farias, P. A. M. J. Am. Chem. Soc. 1996, 118, 7667–7670. (36) Kerman, K.; Ozkan, D.; Kara, P.; Erdem, A.; Meric, B.; Nielsen, P. E.; Ozsoz, M. Electroanalysis 2003, 15, 667–670. (37) Kara, P.; Kerman, K.; Ozkan, D.; Meric, B.; Erdem, A.; Nielsen, P. E.; Ozsoz, M. Electroanalysis 2002, 14, 1685–1690. (38) Ozkan, D.; Kara, P.; Kerman, K.; Meric, B.; Erdem, A.; Jelen, F.; Nielsen, P. E.; Ozsoz, M. Bioelectrochemistry 2002, 58, 119–126. (39) Ozkan, D.; Erdem, A.; Kara, P.; Kerman, K.; Gooding, J. J.; Nielsen, P. E.; Ozsoz, M. Electrochem. Commun. 2002, 4, 796–802. (40) Wang, J.; Nielsen, P. E.; Jiang, M.; Cai, X.; Fernandes, J. R.; Grant, D. H.; Ozsoz, M.; Beglieter, A.; Mowat, M. Anal. Chem. 1997, 69, 5200–5202. (41) Wang, J.; Rivas, G.; Cai, X.; Chicharro, M.; Parrado, C.; Dontha, N.; Begleiter, A.; Mowat, M.; Palecek, E.; Nielsen, P. E. Anal. Chim. Acta 1997, 344, 111–118. (42) Nielsen, P. E. Acc. Chem. Res. 1999, 32, 624–630. (43) Nielsen, P. E. Pure Appl. Chem. 1998, 70, 105–110. (44) Nielsen, P. E.; Haaima, G. Chem. Soc. ReV. 1997, 26, 73–78. (45) Igloi, G. L. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 8562–8567. (46) Kerman, K.; Vestergaard, M.d.; Nagatani, N.; Takamura, Y.; Tamiya, E. Anal. Chem. 2006, 78, 2182–2189. (47) Steichen, M.; Decrem, Y.; Godfroid, E.; Buess-Herman, C. Biosens. Bioelectron. 2007, 22, 2237–2243. (48) Liu, J.; Tian, S.; Nielsen, P. E.; Knoll, W. Chem. Commun. (Cambridge, U.K.) 2005, 2969-2971. (49) Wittung, P.; Kim, S. K.; Buchardt, O.; Nielsen, P.; Norden, B. Nucleic Acids Res. 1994, 22, 5371–7.
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groove.22,50 The binding of C12VC6VC12 in the minor groove was a cooperative process, similar to that described by Armitage et al., who studied the minor groove aggregation of cyanine dye molecules in dsDNA51-53 and PNA-DNA54,55 hybrids. Here we extend that previous work employing C12VC6VC12 to assay the hybridization of PNA to either matched or mismatched DNA targets. We show that binding occurs in the minor groove on matched PNA-DNA hybrids and produces the same unique electrochemical response as was seen when using DNA hybrids. In addition, the electrochemical signal attributed to the minor groove binding of the molecule in the PNA-DNA hybrid is completely attenuated upon exposure of C12VC6VC12 to a PNAmismatched DNA hybrid containing a single altered base. The analysis of dual electrochemical signals indicative of complementary hybridization offers a new approach in the analysis of mismatched DNA segments that is often performed by monitoring the loss of a single electrochemical signal.56,57 Thus, this contribution provides insight into the use of immobilized PNA probes and redox-active minor groove binders for discrimination of complementary and mismatched hybrids.
Experimental Section Materials. All solutions were made with 18 MΩ deionized (DI) water from a Millipore Milli-Q system. Thiolated PNA probes with 15-mer sequence (truncated from a 25-mer sequence used previously)21 were purchased from Applied Biosystems (Bedford, MA). The sequence was HS-Cys-OO-CAC GAC GTT GTA AAA (Cys ) cysteine amino acid residue for thiol (HS) terminus; O ) 8-amino3,6-dioxaoctanoic acid). The PNA was diluted to 5 µM aliquots each with volume of 100 µL in N2-purged DI H2O and stored frozen at -20 °C until use. DNA oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA). The complementary single strand target was a 25-mer with the sequence 5′-CTG GCC GTC GTT TTA CAA CGT CGT G-3′. The mismatched strand had the sequence 5′-CTG GCC GTC GTC _ TTA CAA CGT CGT G-3′ and is referred to as mmDNA. Potassium phosphate and TRIS buffer were purchased from Sigma. 6-Mercapto-1-hexanol (MCH) and sodium dithionite were purchased from Aldrich and used as received. Electrode Preparation and Procedures. The gold electrode (CH Instruments, Austin, TX) used in these experiments had a 2 mm diameter and a surface roughness after polishing of 1.9 ( 0.2, as determined by gold oxide stripping in 0.5 M H2SO4.58,59 The cleaning of the electrode has been described elsewhere.22 Immediately after removal of the electrode from the H2SO4 and drying with N2, it was placed in the 5 µM solution of PNA probe for 15 s followed by rinsing with DI water. This limited exposure time results in adsorption of a small amount of the PNA on the surface. After immobilization of PNA, the electrode was immersed in a 1 mM solution of MCH in deionized water for 30 min, which adsorbs at the remaining Au surface sites and passivates the surface toward nonspecific adsorption of DNA.60 After the MCH treatment, the electrode was rinsed again with DI H2O. Hybridization of the immobilized PNA with the target DNA (50) Tang, X.; Schneider, T. W.; Walker, J. W.; Buttry, D. A. Langmuir 1996, 12, 5921–5933. (51) Garoff, R. A.; Litzinger, E. A.; Connor, R. E.; Fishman, I.; Armitage, B. A. Langmuir 2002, 18, 6330–6337. (52) Wang, M.; Silva, G. L.; Armitage, B. A. J. Am. Chem. Soc. 2000, 122, 9977–9986. (53) Hannah, K. C.; Armitage, B. A. Acc. Chem. Res. 2004, 37, 845–853. (54) Dilek, I.; Madrid, M.; Singh, R.; Urrea, C. P.; Armitage, B. A. J. Am. Chem. Soc. 2005, 127, 3339–3345. (55) Smith, J. O.; Olson, D. A.; Armitage, B. A. J. Am. Chem. Soc. 1999, 121, 2686–2695. (56) Mizuta, M.; Terada, T.; Seio, K.; Sekine, M. Nucleic Acids Symp. Ser. 2004, 48, 237–238. (57) Lee, H. Y.; Park, J. W.; Kawai, T. Electroanalysis 2004, 16, 1999–2002. (58) Brummer, S. B.; Makrides, A. C. J. Electrochem. Soc. 1964, 111, 1122– 1128. (59) Bard, A. J.; Faulkner, L. R. Electrochemical Methods: Fundamentals and Applications, 2nd ed.; John Wiley & Sons: New York, 2001.
Mismatched DNA Hybridization or mmDNA was done by immersing the modified electrode for 2 h in a 50 µM solution of the DNA target strands containing 1.0 M NaCl, 10 mM TRIS buffer, and 1 mM EDTA at pH 7.5 (TE buffer) at room temperature followed by rinsing with TE buffer solution. When preparing solutions of C12VC6VC12, a stock solution in Millipore water was made by heating to 35 °C in order to rapidly solubilize the compound. Diviologen solutions for the electrochemical experiments were made by addition of small amounts of the stock solution to the supporting electrolyte solution. The surfaces with immobilized PNA or PNA-DNA were exposed to solutions containing low (ca. µM) concentrations of the diviologen derivatives. Typically, the PNA-DNA surfaces were produced by hybridizing the complementary strand with the same surface used for the PNA evaluation, thus providing for a direct comparison at identical surfaces. This exposure resulted in preconcentration of C12VC6VC12 at the modified surface due to interaction with the immobilized PNA or PNA-DNA hybrids. Electrochemical assay of the surface population of viologen groups was done directly in the solution containing the diviologen in the electrolyte buffer (4 mL volume, 5 mM TRIS, 10 mM NaCl, pH 7.4). For cyclic voltammetric assay experiments the electrochemical response from the dissolved diviologens was negligible compared to that from the surface population at the concentrations and scan rates employed. For chronocoulometric assays, Anson plots were used to separate the surface response from the solution phase response.21 DNA Surface Coverage. An electrochemical DNA assay20,21,61 based on electrostatic binding of Ru(NH3)63+ was used to quantitate the number of DNA phosphate groups immobilized on the surface after PNA-DNA hybridization. This allows calculation of the amount of immobilized DNA and, based on the extent of hybridization from analogous ssDNA surfaces,22 allows one to determine the amount of immobilized PNA probes at the electrode surface. The amount of complementary target DNA present on the PNA-DNA surface was calculated to be (5.0 ( 0.50) × 10-12 mol cm-2. Assuming 90% hybridization, the surface coverage, Γ, of PNA probes on the electrode surface was calculated to be (5.5 ( 0.55) × 10-12 mol cm-2. Likewise, Γ for the amount of mmDNA remaining after the TE rinse was calculated to be (1.8 ( 0.45) × 10-12 mol cm-2. At this surface coverage, the average separation distance between DNA molecules on the surface is ∼6.5 nm, which is longer than the length of the 15-mer PNA probe. However, some, limited interaction may be possible between the longer, unhybridized sections of target DNA strands. UV-vis Spectroscopy. The PNA, PNA-DNA, or PNA-mmDNA hybrids were diluted to a concentration of 1.35 µM in 1 mL of 50 mM TRIS, 10 mM NaCl, pH 8.0 in a plastic cuvette. C12VC6VC12 was added in an appropriate ratio along with 1.15 mM sodium dithionite to reduce the viologen groups to their cation radical state. The spectra were recorded immediately after the addition of dithionite. Synthesis. C12VC6VC12 was synthesized following our previously reported procedure.22 Instrumentation. Cyclic voltammetry and chronocoulometry were done using a Gamry Instruments FAS1 Femtostat controlled with PHE200 Physical Electrochemistry Software (Warminster, PA). The reference electrode was a Ag/AgCl (saturated KCl) electrode manufactured locally. The counter electrode was a platinum wire. All solutions were aggressively purged with N2 prior to electrochemical experiments. UV-vis measurements were made with a Hewlett-Packard 8452A diode array spectrophotometer. Origin 7.0 graphing software was used to analyze cyclic voltammograms to extract surface populations of the various interfacial species. A standard multiple curve fitting algorithm was used.
Results Chronocoulometric Analysis. Uptake of C12VC6VC12 onto the different PNA-modified Au electrodes was determined using (60) Steel, A. B.; Lecicky, R. L.; Herne, T. M.; Tarlov, M. J. Biophys. J. 2000, 79, 975–981. (61) Tarlov, M. J.; Steel, A. B. DNA Based Sensors. In Biomolecular Films: Design, Function, and Applications; Rusling, J. F., Ed.; Marcel Dekker: New York, 2003; pp 545-607.
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Figure 1. Chronocoulometry-derived adsorption isotherms for C12VC6VC12 binding to a Au electrode modified with (a) PNA-DNA, (b) PNA-mismatch DNA, and (c) PNA only. The responses were corrected after subtraction of the response from a MCH-Au modified surface. The colored lines represent the Frumkin isotherm fits for each data set. Conditions: initial voltage 0.1 V, final voltage -0.7 V, t ) 0.1 s, 5 mM Tris 10 mM NaCl, pH 7.4, N2 purge.
a previously described chronocoulometric method in which the potential is stepped over the wave for the one electron reduction of the dicationic viologen group to the cation radical state, consuming two electrons per diviologen.21 This provides surface coverage and thermodynamic binding data but offers little information on the mode of binding to the immobilized hybrids.22 Figure 1 shows the adsorption isotherms of C12VC6VC12 at the different modified surfaces. The curves show C12VC6VC12 uptake on the PNA-DNA, PNA-mmDNA, and PNA only surfaces after subtraction of the C12VC6VC12 response on MCH. As can be seen, there is a small amount of additional nonspecific binding of C12VC6VC12 at the MCH/PNA surface compared to the MCH control. Previously, the 4+ charge on the diviologen molecule was shown to drive binding to DNA surfaces based on electrostatic interactions.22 Therefore, the lack of significant binding of the diviologen to the unhybridized PNA compared to single-stranded DNA is an expected result of the neutrality of the PNA. Weak interactions between the hydrophobic PNA moieties and the hydrophobic portions of C12VC6VC12 presumably lead to the small increase in binding at the MCH/PNA surface compared to the MCH surface. Figure 1a,b shows the isotherms upon C12VC6VC12 exposure to the PNA-DNA hybrid and PNA-mmDNA. The hybrid surface was formed by exposure of the PNA probe surface to a 25-mer ssDNA target containing the complementary 15-mer PNA probe sequence. The mismatch hybrid surface was formed via exposure of the PNA probe to a target containing a mid sequence T f C (the mismatch is located one base from the C terminus of the PNA probe). The isotherms show that there is an increase of diviologen bound to both of these surfaces compared to the PNA surface, which reflects the attraction of C12VC6VC12 to the anionic DNA oligomer. In addition, the binding isotherms of C12VC6VC12 on the complementary PNA-DNA hybrid and PNA probe surface shows upward curvature at low solution concentrations (0-3 µM). This denotes a cooperative binding process, which can be fit with a Frumkin model:
KC )
θ -2g′θ e 1-θ
where K is the binding constant (M-1), C is the solution concentration of C12VC6VC12 (M), θ is the normalized surface
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Table 1. C12VC6VC12 Binding to PNA-Modified Au Surfaces Au electrode
Γsat (10-12 mol/cm2)a
g′
g (1011 kJ cm2/mol2)
K (105 M-1)
∆G (kJ/mol)
PNA PNA-DNA PNA-mmDNA PNA-DNA ”B” population
3.32((0.4) 25.1((3) 11.0((3) 10.9((1)
1.4((0.2) 2.0((0.2) 0.30((0.06) 5((0.5)
10.4 1.97 0.680 56
7.0((0.3) 0.480((0.1) 8.0((0.3) 0.394((0.05)
-27.6((0.3) -26.7((0.2) -33.7((0.1) -26.2((0.6)
a
Corrected for MCH contribution.
coverage of C12VC6VC12 (Γ/Γsat), and unitless g′ ) 2gΓsat/RT, where g is the interaction parameter, which can be thought of as a change in the binding free energy as the surface coverage changes (J mol-1 per mol cm-2).59 Positive values of g′ indicate attractive interactions between bound diviologen molecules, a signature of cooperative binding. The origin of this cooperative binding is discussed in more detail below. The data show an increased amount of diviologen binding to the complementary PNA-DNA hybrid compared to that at the PNA-mmDNA hybrid, presumably because there is more hybridization in the complementary case than in the mismatched case. Thermodynamic data for diviologen binding to each of the modified surfaces were obtained from the respective isotherm fits (lines in Figure 1) and are summarized in Table 1 (the data labeled PNA-DNA “B” population are discussed further below). The binding constants for C12VC6VC12 to the PNA, PNA-DNA, and PNA-mmDNA hybrids are in the range of ∼(0.5-5) × 105 M-1, with similar free energies of binding (∆G) on all surfaces (approximately -30 kJ mol-1). These values are in the range of those previously reported for diviologen binding to immobilized 25-mer DNA-DNA complementary hybrids.22 The data show slightly different binding of the diviologen to the various surfaces. The largest binding constant is observed for the PNA-mismatch DNA hybrid, while the smallest is observed for the PNA-DNA hybrid. This is likely because the mismatch distorts the duplex, exposing the hydrophobic bases. This may enhance the hydrophobic interaction between the diviologen and the PNA-mmDNA hybrid compared to that between the diviologen and the complementary hybrid. The data also show considerable cooperativity for diviologen binding to the PNA-DNA hybrid, with a g′ value approaching 2.0. As discussed previously, cooperativity in the isotherm is consistent with minor groove binding of the diviologen.22 Cyclic Voltammetric Analysis. A clearer picture of the interaction of the diviologen with the different surfaces is provided from cyclic voltammetry. Figure 2a-c shows the evolution of the cyclic voltammetric response at each modified surface as the concentration of the diviologen in solution is increased over the range 50 nM to 8 µM. In this range of concentration and scan
Figure 2. Cyclic voltammograms of C12VC6VC12 at 50 nM (red), 0.3 µM (navy), 1 µM (green), 2 µM (blue), 3 µM (turquoise), 4 µM (fuschia), 5 µM (yellow), 6 µM (olive), 7 µM (purple), and 8 µM (brown) in the presence of (a) PNA only, (b) PNA-mmDNA, and (c) PNA-DNA hybrids. Points A and B in (c) denote the two bound diviologen populations to PNA-DNA hybrids. CV conditions: 0 to -0.75 V vs Ag/AgCl; 100 mV s-1, 5 mM Tris, 10 mM NaCl, pH 7.4, N2 purge.
rate only the surface immobilized diviologens are observed. All of the surfaces preconcentrate the diviologen to some extent, as can be seen by the increase of the wave for the reversible oneelectron reduction of the viologen group to the cation radical state. Parts a and b of Figure 2 show the responses at PNA and PNA-mmDNA surfaces, respectively. The formal potential for the one-electron reduction of the viologen groups in the diviologen molecule at these two surfaces is observed at -0.48 V vs Ag/ AgCl. This type of electrochemical behavior was previously seen for this molecule on analogous ssDNA surfaces.22 The peak shapes are approximately symmetric, and the peak current is directly proportional to scan rate (data not shown), indicative of surface-confined electrochemistry.59 The only significant difference between the two cyclic voltammograms in Figure 2a,b is the higher saturation surface coverage for the PNA-mmDNA surface, presumably because of the electrostatic attraction between the diviologens and the anionic DNA chains. Binding to the PNA surface is likely driven by hydrophobic interactions. Figure 2c shows the C12VC6VC12 voltammetric response in the presence of complementary PNA-DNA hybrids. The wave shape of the CVs is qualitatively different than those in Figure 2a,b, containing a double-wave shape emerging at solution concentrations as low as 1 µM. The first wave occurs at -0.52 V vs Ag/AgCl, and a second wave also is observed, shifted 100 mV negatively. The double-wave shape is indicative of different diviologen populations in multiple binding sites on cDNA hybrids and was described previously.22 For convenience, the species reduced at -0.62 V vs Ag/AgCl is referred to as the “B” population, and that reduced at -0.52 V vs Ag/AgCl is referred to as the “A” population. The significant negative shift in potential of the B peak denotes that this surface bound population is more stable in the oxidized form in the presence of hybrids vs the A population. In order to extract the isotherms of the A and B populations on the PNA-DNA hybrids, a multiple curve fitting algorithm was applied to baseline subtracted reduction scans of the CVs in Figure 2c. The reduction scans were used as they provided better background subtractions. A sample baseline subtracted CV along with the generated fits of the multiple peaks is shown in Figure S1 (Supporting Information). Isotherms generated from the integration of these two waves vs C12VC6VC12 solution concentration are shown in Figure 3. Figure 3a shows the contribution from the A wave (-0.52 V vs Ag/AgCl). The uptake of this population occurs relatively slowly at lower solution concentrations, accelerates at solution concentrations of 3 µM, and becomes less dependent on concentration at the higher solution concentrations. While there is no clear evidence for saturation in this isotherm, there is a suggestion of a transition in the isotherm at higher concentrations. Note that, given the number of phosphate groups present at this surface, the saturation coverage of C12VC6VC12 based on electrostatic considerations would be 3.1 × 10-11 mol cm-2. Figure 3b shows the response of the uptake of the B population. The behavior of this bound population is very similar to that seen when diviologen binding to cDNA-DNA hybrids was examined.22 The strong upward curvature in the isotherm is indicative of a strongly cooperative
Mismatched DNA Hybridization
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that are not interacting strongly with each other). In contrast, the λmax in the presence of the complementary PNA-DNA hybrid is observed at 535 nm. This blue shift is typical of face-to-face π-dimerization of the viologen cation radical ring systems50 and suggests that this type of dimerization takes place in the presence of PNA-DNA hybrids, just as was observed for cDNA-DNA hybrids.22
Discussion Figure 3. CV integration generated adsorption isotherms demonstrating the binding of (a) the A population and (b) the B population to the PNA-DNA complementary hybrids. The red line is the Frumkin fit for the B population adsorption.
Figure 4. Visible absorbance spectra of singly reduced C12VC6VC12 at a ratio of 2:1 in the presence of PNA only (green), PNA-mmDNA (red), and PNA-DNA complementary hybrids (blue). Conditions: 1.35 µM PNA (hybrid), 2.7 µM C12VC6VC12, 1.15 mM NaS2O4, 50 mM Tris, 10 mM NaCl, pH 8.0, N2 purge.
binding process. Indeed, the cooperativity constant of g′ ) 5 based on the Frumkin curve fit (red line in Figure 3b) is much higher than what was seen in the chronocoulometric isotherms (Figure 1) and what was reported for binding to ssDNA probes (g′ ) 3.65).22 The saturation of this molecule occurs at ∼4 µM, which corresponds to a surface coverage of C12VC6VC12 of 1.1 × 10-11 mol cm-2 (6.6 × 1012 molecules cm-2). Based on the Ru(NH3)63+ assay, the amount of PNA-DNA hybrids present on the electrode surface was determined to be 5.0 × 10-12 mol/ cm2 (3.0 × 1012 hybrids cm-2). Therefore, upon saturation of the B population, approximately two diviologen molecules are bound to each PNA-DNA hybrid. Thermodynamic information for the C12VC6VC12 B population bound on PNA-DNA hybrids is also summarized in Table 1, labeled as PNA-DNA “B” population. The 2:1 ratio of diviologen molecules to hybrids at saturation coverage of the B population is different from the 4:1 ratio of diviologen molecules to hybrids for binding to cDNA-DNA hybrids reported previously,22 for reasons discussed below. Visible Absorbance Spectroscopy. Solution phase visible absorbance spectra of chemically reduced C12VC6VC12 were obtained to clarify the nature of its interactions with PNA, PNA-mmDNA hybrids, and PNA-DNA hybrids. Dithionite reduction was used to produce only the one-electron-reduced cation radical state of the viologen groups in the diviologen compound, as previously described.22 Figure 4 shows absorption spectra in the visible region for reduced C12VC6VC12 in the presence of PNA, PNA-mmDNA, and PNA-DNA. In the presence of PNA and PNA-mmDNA, the viologen cation radicals in the diviologens show absorption maxima at 555 and 553 nm, respectively. These wavelengths are characteristic of isolated, monomeric viologen cation radicals (i.e., cation radicals
The data described above show some similarities to those in a previous report in which interaction of the C12VC6VC12 molecule with ssDNA and complementary dsDNA hybrids was explored but exhibit also exhibit some interesting differences.22 The previous results pointed unequivocally to the B population cyclic voltammetric peak as being caused by the formation of faceto-face π-dimers in the minor groove when the viologen groups in the bound diviologens were reduced by one electron each. This dimerization is similar to the aggregation of cyanine dyes in DNA hybrid minor grooves.51,52 We assign the B population peak to the same diviologen dimerization in the PNA-DNA hybrid case. In this case, the geometrical requirements of π-dimerization and the geometry of the PNA-DNA minor groove are responsible for the ability of the diviologen to electrochemically distinguish between PNA-DNA complementary hybrids and mismatched hybrids. The ability of the diviologen to differentiate PNA-DNA complements and mismatched hybrids can be understood by comparing dsDNA to PNA-DNA hybrids. PNA-DNA duplexes have a slightly different structure than dsDNA owing to the more rigid 2-(aminoethyl)glycine backbone of the PNA. The duplex length is altered slightly by a larger base pair separation.42,43,62 Also, the minor groove dimensions are changed compared to the dsDNA minor groove, primarily in the y-axis, making the groove much narrower.62 This narrowed groove dimension is responsible for cooperativity in the binding of some compounds in the minor groove. Previous reports by Armitage and co-workers have detailed the binding of planar cyanine dyes in PNA hybrids.54,55 They found that aggregates of this compound and its analogues bind in the minor grooves of not only DNA but also antiparallel PNA-DNA hybrids.55 They argued that binding was cooperative based on the widening of the groove after the first dye molecule initially binds, which facilitates binding of additional molecules in the groove as aggregates.55 We believe that similar factors are responsible for the cooperativity for diviologen binding to PNA-DNA hybrids. It is known that the minor groove geometry in PNA-DNA hybrids is significantly affected by single-base mismatches.55,63 As a consequence, the spectroscopically detected aggregation of cyanine dyes in the minor groove is sensitive to mismatches.55 We believe the π-dimerization of the diviologen has a similar sensitivity, so that π-dimerization is not possible in the minor groove of a PNA-mmDNA hybrid. Even though there is no minor groove binding in the PNA-mmDNA case (Figure 2b), there still is significant binding of the diviologen to the PNA-mmDNA surface. This is likely due to the electrostatic binding of the diviologen to DNA in the mismatched hybrid. However, the complete disappearance of the B population peak reflects a significant change in the binding location for the diviologen, which must be on the exterior of the hybrid as opposed to in the minor groove. The UV-vis results (62) Eriksson, M.; Nielsen, P. E. Nat. Struct. Biol. 1996, 3, 410–413. (63) Menchise, V.; De Simone, G.; Tedeschi, T.; Corradini, R.; Sforza, S.; Marchelli, R.; Capasso, D.; Saviano, M.; Pedone, C. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 12021–12026.
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in Figure 4 are also consistent with this in that very little faceto-face π-dimer formation exists on mismatched hybrids based on the negligible blue shift in the spectrum. Another interesting difference between the binding of the diviologen to dsDNA is that saturation binding in the present case occurs for a ratio of C12VC6VC12/hybrid of 2:1 while in our previous study the saturation ratio was 4:1.22 The 2:1 versus 4:1 ratio is due to the length of the PNA probe, which is only 15 nucleobases vs the 25-mer ssDNA used in the previous case. Based on the length and pitch of the 15-mer PNA used here, ∼65 Å of available minor groove binding length is available to the diviologen. With face-to-face dimers in the minor groove, this length can accommodate two fully elongated C12VC6VC12 molecules (50 Å in length from terminal methyl to terminal methyl). In the previous case, the 100 Å length of the dsDNA minor groove could accommodate four face-to-face dimerized diviologens.22 Thus, the difference in binding ratio at saturation is completely explained by the length of the minor groove in the two cases. The ability of C12VC6VC12 to bind in intact minor grooves of PNA-DNA hybrids is unique in the class of electroactive compounds used to report on hybridization. Further, very few literature reports describe molecules designed specifically to exploit the structural features of PNA-DNA hybrids. A key reason for this is the difference in PNA-DNA and dsDNA duplex morphology including altered groove dimensions and increased internucleobase distances.43,62 These changes attenuate the binding of many traditional intercalators and minor groove binders
HVastkoVs and Buttry
exploited to detect dsDNA.49 This results in PNA biosensors employing electrochemical transduction platforms based on relatiVe redox signal changes via monitoring of guanine oxidation36,37 or the binding of cationic transition metal complexes.35,40,41,46,47 In contrast, the presence or absence of the double-wave CV response seen here provides a distinct, new signal that gives a definitive indication for the presence of a genetic segment of interest. The ability to differentiate a complementary or mismatched 25 bp target with a terminal mismatch lends confidence in the ability of the platform to detect damage to DNA segments of interest over time or detect damaged gene fragments. Indeed, work in this area is currently underway in our laboratory. In summary, we have described a Au electrode immobilized PNA probe platform that uniquely distinguishes between mismatched and cDNA targets based on the minor groove binding of singly reduced cation radical viologen groups C12VC6VC12. This ability to qualitatively detect single-base mismatches opens new possibilities in the field of electrochemical DNA hybridization detection. Acknowledgment. E.H. thanks Drs. Reinaldo and Fernanda Bazito for their helpful organic synthesis guidance. Supporting Information Available: Two additional images: baseline subtracted multiple curve fit CV and 4 mM PNA-DNA and PNA-mm DNA CV comparison. This material is available free of charge via the Internet at http://pubs.acs.org. LA803284R
