DNA conductivity in DNA sensors

6.5.1. DNA conductivity

The idea that dsDNA may function as a conduit for fast electron transport along the axis of its base-pair stack was advanced more than 40 years ago (Porath et al., 2004 and references therein). But later low-temperature experiments suggested charge migration within the frozen water layer surrounding the DNA double helix (Warman et al, 1996). In the recent decade, investigations of the DNA conductivity became a hot topic because of its relevance for a number of biological processes, such as those involved in mutagenesis and cancer, and for molecular electronic. The literature on the DNA conductivity was reviewed in several articles, e.g. (Barbara and Olson, 1999; Grinstaff, 1999) and in 2004 a special volume of Topics in Current Chemistry (2004) was devoted to this topic. Here, only a brief summary will be presented with special attention to application of DNA charge transport in electrochemical sensors for DNA hybridization.

Solution chemistry experiments on a number of short DNA molecules indicated high charge transfer rates between a donor and acceptor, which were located at distant DNA sites (Porath et al., 2004). Several mechanisms were proposed for DNA-mediated charge transport, in dependence on the structural aspects of the system under investigation, including the DNA base content and/ or sequence. These advances stimulated interest in DNA for nanoelectronics and led to a set of direct electrical transport measurements. A number of ingenious experiments with single molecules, bundles and networks were performed showing that it is possible to transport charge carriers along DNA molecules. After some initial controversial reports on the conductance of DNA devices, recent results indicated that short DNA molecules were capable of transporting charge carriers, similarly to DNA bundles and DNA networks. On the other hand it was shown that transport through single DNA molecules, longer than 40 nm was blocked. This might be due to the DNA interaction with the surface which may induce defects in the DNA molecules disturbing the electronic structure of dsDNA (including the DNA surface denaturation, see Section 5) and blocking the charge transport. The question whether DNA has properties of a metal, semiconductor or an insulator was frequently asked. This terminology originates from solid-state physics. It is, however, questionable whether such terminology can describe adequately the orbital energetics and the electronic transport through one-dimensional soft polymer, such as DNA, that is formed of a large number of sequentional segments. A large number of junctions and phase-coherent "islands" may influence the transport mechanism along the molecule. In some cases such junctions can constitute a rate-limiting step for the transport. In spite of the outstanding progress in controlling the self-assembly of DNA on electrodes (Section 6.1.2), unanimously accepted explanation of the mechanisms responsible for the charge mobility through the dsDNA structure is not yet available. On the other hand, charge transfer in self-assemblies of thiolated dsODNs was exploited in development of the electrochemical DNA sensors (Drummond et al, 2003).

6.5.2. Charge transfer in DNA sensors

Electrochemistry at chemically modifed surfaces has been applied to investigate charge transport in various media including thioalkanes and proteins (Armstrong et al, 2000; Chi et al, 2001; Creager et al, 1999; Finklea, 1996; Immoos et al., 2004c; Munge et al., 2003; Nahir and Bowden, 1996; Niki et al., 2003; Sachs et al., 1997). Barton and coworkers reported self-assembly of 15-20 base pair dsODNs (containing different nucleotide sequences) covalently attached to gold surfaces via thiol tethers and demonstrated unique charge transfer characteristics through this assemblies (Drummond et al., 2003). They developed electrochemical assays for DNA hybridization, including detection of point mutations as well as for DNA damage and DNA-protein interactions. Typically, DNA duplexes were prepared in solution and self-assembled on gold electrodes. The electrode was then treated with planar redox active molecules, such as methylene blue (MB) which was non-covalently bound (intercalated) to

Fig. 29. Scheme of electrochemical assay for mismatches through DNA-mediated charge transport. On the right is shown an electrode modified with well-matched duplex DNA. Current flows through the well-stacked DNA to reduce methylene blue (MB+) intercalated near the top of the film, to leucomethylene blue (LB). LB goes on to reduce ferricyanide in solution, thereby regenerating MB + catalytically, leading to an amplification of the hybridization signal. In the case of a DNA film containing mismatched duplexes (left), current flow through the DNA duplex is attenuated, MB + is not reduced, and the catalytic signal is lost. Adapted from Drummond et al. (2003). Copyright 2003, with permission from the author.

Fig. 29. Scheme of electrochemical assay for mismatches through DNA-mediated charge transport. On the right is shown an electrode modified with well-matched duplex DNA. Current flows through the well-stacked DNA to reduce methylene blue (MB+) intercalated near the top of the film, to leucomethylene blue (LB). LB goes on to reduce ferricyanide in solution, thereby regenerating MB + catalytically, leading to an amplification of the hybridization signal. In the case of a DNA film containing mismatched duplexes (left), current flow through the DNA duplex is attenuated, MB + is not reduced, and the catalytic signal is lost. Adapted from Drummond et al. (2003). Copyright 2003, with permission from the author.

the densely-packed DNA-modified gold electrode. It was shown that these in-tercalators bound near the solution accessible interface of the densely packed DNA-modified surfaces eliminating the need for covalent binding (Boon et al., 2000, 2002a, b, 2003; Kelley et al., 1999b; Sam et al., 2001). The reduction of the intercalator at the top of the film through a DNA mediated reaction reflected base-pair stacking within the film (Figure 29). With intact stacking MB was efficiently reduced at the DNA film but in presence of an intervening mismatch or other duplex perturbation attenuation of MB reduction was observed (Boon et al., 2000, 2002a, b; Kelley et al., 1999a, b). In some cases, the MB signal was amplified through an electrocatalytic cycle. MB served as a catalyst for the reduction of ferricyanide diffusing in solution outside of the DNA film (Boon et al., 2000, 2002b, Kelley et al., 1999a). It was shown that both the direct and catalytic reduction of MB took place via charge transfer through the DNA base stack (Boon et al., 2003; Kelley et al., 1997a). Thus DNA mediated charge transfer electrochemistry represented a sensitive probe of nucleic acid structure and base stacking. Even minor perturbations in stacking diminished the MB reduction signal (Boon et al., 2002b; Kelley et al., 1999a). Electrochemical reduction of DNA intercalators was used to study DNA-protein interactions (Boon et al., 2002a) and hybridization of antisense oligonucleotides (Boon et al., 2002b). Charge transfer in right-handed A- and B-form DNA double helices and in left-handed Z-DNA was investigated (Boon and Barton, 2003). The A-DNA was examined in the context of a DNA/RNA hybrid duplex and Z-DNA at high Mg2+ concentrations in duplexes containing methylated cytosine in d(CG)n sequences. Efficient charge transfer was detected in both A- and B-DNA using MB reduction as a probe. In Z-DNA films lower level of MB reduction was observed. Less efficient but not completely attenuated charge transfer in Z-DNA can be due to different base stacking related to different structural parameters and particularly to the alternating syn-anti-sugar conformation in Z-DNA (in difference to A- and B-DNA there is no intra-strand stacking in Z-DNA), etc.

Alternatively, the intercalating probe was site-specifically coupled to the HS-ODN before the self-assembly to control precisely the location of the inter-calator (Kelley et al., 1999a). This technique was used to probe the distance dependence of the charge transfer by preparing a series of DNA-modified electrodes in which the through helix distance from the intercalator (daunomycin, DM) to the gold surface spanned over 4.5 nm. It was found that the electrochemical response of the intercalated DM was not dependent on its location in the DNA helix (Kelley et al., 1999a). Considering the very shallow distance dependence of the charge transfer in solution (Boon and Barton, 2002; Hall et al., 1996; Kelley et al., 1997b; Nunez et al., 1999; Wan et al., 1999; Yoo et al., 2003) and striking dependence of the presence of a single-base mismatch in the duplex, the role of the length of the linker was tested (Drummond et al., 2004). A homologous series of DM-labeled ODN assemblies featuring thiol-terminated linkers possessing different numbers (n) of methylene units conjugates (with n ranging from 4 to 9) were constructed. The resulting ODN-DM molecules were self-assembled on gold electrodes with excess Mg2+ to obtain dense monolayers (surface coverages ranged from 30 to 75pmol/cm2). Irrespective of linker lengths a chemically reversible reduction of DM was observed at about —0.6 V (vs. Ag/ AgCl 1 M KCl). On the other hand, the intensity of the electrochemical response decreased with increasing linker length. It was concluded that in the time scale of the CV experiments (scan rates ~1V/s) the ODN-DM conjugates behaved as discrete redox-active entities, with electrochemical responses independent of the DM intercalation site in a good agreement with results of the scanning tunneling microscopy (STM) studies of the self-assemblies on gold (Ceres and Barton, 2003). Irrespective of the mechanism of the charge transfer through the ODN assembly, charge transfer through the s-bonded linker followed semiclassical superexchange theory. Thus, when the linker lengths and the DM positions varied the charge transfer through the s-bonded linker was the rate-limiting step (Drummond et al., 2004).

The conclusions of Barton et al. (Boon and Barton, 2002; Drummond et al., 2004; Kelley et al., 1999a; Yoo et al., 2003) based on voltammetric responses of their systems were supported by the results of investigation of thiol-modified ODN films on gold surfaces using electrochemical in situ scanning tunnelling microscopy (Ceres and Barton, 2003). This technique revealed effective charge transport on gold (under conditions close to physiological) depending upon ODN orientation and integrity of base-pair stacking. Base mismatches behaved as electronic perturbations exerting strong effects on the ODN conductivity in agreement with electrochemical (Boon et al., 2000), photophysical (Kelley et al., 1997b) and biochemical (Bhattacharya and Barton, 2001) studies. dsDNA was suggested (Ceres and Barton, 2003) as a promising candidate in molecular electronics under the conditions that the orbitals could efficiently overlap with the electronic states and the environment did not disturb the DNA double helical structure, forming non-native poorly stacked DNA conformations.

DNA-mediated charge transport through DNA self-assemblies at gold electrodes was exploited to examine the effect of the presence of base analogs and DNA damage products in ODN duplexes (Boal and Barton, 2005). General trends in how base modifications affect charge transfer efficiency were found. A decrease in the charge transfer efficiency was caused by: (a) modifications of the Watson-Crick hydrogen bonding system or addition of steric bulk; (b) base structure modification inducing conformational changes such as burying of hydrophilic groups within the DNA double helix. On contrary addition or subtraction of methyl groups, not disrupting hydrogen bonding interactions, did not show a large effect on charge transfer efficiency. No simple correlation between the charge transfer efficiency and DNA melting temperatures was found. It was suggested that the sensitive detection methodology (monitoring the electrocatalytic reduction of MB) might be useful as a possible damage detection for DNA repair enzymes and in diagnostic applications.

The results of Barton's group showed an interesting and convincing picture of charge transfer in relatively short DNA double helices (Drummond et al., 2003). It has been shown in many experiments that efficient charge transfer reactions can occur in short well-stacked ODNs self-assembled at the gold surfaces (Bhattacharya and Barton, 2001; Boal and Barton, 2005; Boon and Barton, 2002, 2003; Boon et al., 2000, 2002a, b, 2003; Ceres and Barton, 2003; Drummond et al., 2003, 2004; Hall et al., 1996; Kelley et al., 1997a, b, 1999a, b, 1998; Nunez et al., 1999; Sam et al., 2001; Wan et al., 1999; Yoo et al., 2003). Recently, several papers have been published which do not show any charge transfer through similar ODN helices under different experimental conditions (Anne et al., 2003; Fan et al., 2003; Immoos et al., 2004a, b; Mao et al., 2003). These papers will be briefly summarized in the following section where we shall return to the problem of the charge transfer in DNA.

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