For the AQ probe, DNA-mediated electrochemistry is found with an acetylene linker on uridine, but not with an alkyl chain at the 5' end of the oligonucleotide. Interrupting the conjugation of the ferrocene tag with the base pair stack results in an almost complete loss of the electrochemical signal. A well-coupled ferrocene-uridine derivative showed a greater electrochemical signal at the top of the DNA monolayer than in the immediate vicinity of the surface (29).
It was suggested that elastic bending of 5'-ferrocene-labeled DNA was the cause of the observed electrochemical signals. It was concluded that DNA bending could explain the electrochemistry of all DNA-bound probes. The reported observations for the AQ and TEMPO probes make them particularly suitable for establishing a correlation between the coupling connecting the probe to the DNA and the effectiveness of the probe in assays based on DNA-mediated electrochemistry.
On DNA-modified Au, we study the difference between the AQ moiety linked to the base pair stack via an acetylenic linker and AQ linked via an alkyl chain to the 5' end of the DNA. Synthesis of ethylenediamine TEMPO-modified DNA was performed according to a protocol adapted from the literature (42). A ~0.5 M aqueous solution of ethylenediamine TEMPO was added to the 5-bromouridine-modified DNA on the solid support, and the resulting suspension was incubated at 60 ºC overnight.
The coupling of the 2-iodoanthraquinone to the ethynyluracil-modified DNA was performed while the DNA was still attached to a solid controlled-pore glass (CPG) support.
Results
The acetylene bond and the 3,4-dehydro-TEMPO probe were designed to allow efficient coupling of the probe into the base pair stack, while the alkane bond was intended to electronically isolate the probe from the base pair stack. Several AQ-modified DNA sequences of 14–17 base pairs in length were prepared, obtaining similar results. Experiments using TEMPO-modified DNA take advantage of the extended potential range offered by HOPG.
A reversible electrochemical signal is found at -301 (±7) mV versus NHE for AQ-modified DNA on Au, and the signal is stable for a minimum of two hundred scans under anaerobic conditions. As expected for a surface-bound species, a plot of peak current versus scan rate is linear for the DNA-bound AQ as shown in Figure 5.3 (50), and the electron transfer rate was estimated to be ca. 30 pp. -1. The DNA sequence used for each electrochemical marker is shown below the structures of the markers.
The location of the mismatch is underlined and the location of the electrochemical probe is indicated with uracil (U). Voltammograms for DNA modified with acetylene-linked AQ (top left), acetylene-linked 3,4-dehydro-TEMPO (top right), alkane-linked AQ (bottom left), and alkane-linked TEMPO ( bottom right ) are shown. The signal potential observed for DNA modified with 3,4-dehydro-TEMPO is similar to that previously reported for the free label in solution (40).
As expected, controls run with well-matched, unmodified DNA reveal no electrochemical signals at the reported potentials for 3,4-dehydro-TEMPO and AQ-modified DNA. The introduction of a mismatch between the two marks causes a significant attenuation of the electrochemical signal (Figure 5.2 and Table 5.1). Notably, the introduction of a CA mismatch across the 3,4-dehydro-TEMPO probe has little effect on the magnitude of the peak current (Figure 5.4).
A small signal centered at mV compared to NHE, the reversibility of which cannot be assessed, is observed for AQ-modified DNA on Au. The sequence was pyrene- (CH2)3CONH(CH2)6NHCO-5'-CTA CAG TCG T-3', where the italic base indicates the TEMPO location and the bold base indicates the mismatch location. Unlike AQ, the magnitude of the TEMPO signal only decreases by a factor of 4 when changing linkage from acetylene to alkane; the peak current drops from nA for acetylene-bound 3,4-dehydro-TEMPO to 60 (±20) nA for ethylenediamine-bound TEMPO.
As with AQ, the smaller size and instability of the TEMPO signal make it difficult to plot peak current as a function of scan rate. In contrast to what was observed for the acetylene-linked probes, the introduction of a mismatch under the alkane-linked AQ and TEMPO labels does not lead to a significant reduction of the electrochemical signals (Figure 5.2).
Discussion
A clear trend emerges from these values: coupling that allows conjugation of the probe into the π stack leads to larger redox signals and more effective electrochemistry. However, at positive voltages, direct contact of the probe with the surface is possible if sufficient space is available around each duplex. The current densities obtained for the alkane-coupled TEMPO and AQ labels are much smaller than those of the acetylene-coupled probes.
AQ is reduced at negative potentials where the DNA bounces off the surface, making direct CT probe reduction from the surface less likely (53, 56). These values are similar to the theoretical maximum surface coverage of a DNA monolayer. Covalently bound daunomycin is particularly tightly bound to the base pair stack and raises the melting temperature of the duplex by 20 ºC upon cross-linking (35, 36).
On the other hand, the bulky AQ unit only increases the melting temperature of the DNA duplex by 4 ºC compared to unmodified DNA. Large electrochemical signals arise, and perturbations within the base pair stack can be easily detected. When the probes are isolated from the base pair stack by a σ-bonded alkane linkage, the yield of DNA-mediated electrochemistry is low.
Direct interaction of the diaminoethane-coupled TEMPO probe with the surface is shown on the left, and DNA-mediated electrochemistry with the well-coupled probe is shown on the right. These results can now be used to reexamine some of the data obtained from long-range oxidative damage experiments on DNA. Consequently, solution studies with covalently linked anthraquinone provide a particularly interesting example of the necessity of coupling in the DNA base pair stack.
In addition, a direct comparison of the ability of different photooxidants to promote DNA-mediated CT showed that even changing the length of the linker connecting the anthraquinone had a significant effect on the yield of oxidative damage. Therefore, it is important to note that DNA-CT reveals the properties not only of the DNA sequence used, but also of the redox probe. Consideration of the intrinsic sensitivity of DNA CT to the coupling of the redox probe to the DNA base pair stack can also be extended to resolution studies of protein/DNA interactions.
Implications
On the other hand, when the TEMPO probe is connected with a saturated coupling, smaller EPR and electrochemical signals result. surface-to-probe charge transfer, although useful in other contexts, does not allow the DNA base pair stack to be interrogated. Indeed, the impressive sensitivity of DNA CT chemistry enables the transduction of small structural changes into large electrochemical differences. As observed in the current study, the coupling directly controls the electrochemical properties of the probe.
For electrochemical tests to be successful, it is crucial to adapt the probe to the desired application. 2005) Charge Transfer in DNA: From Mechanism to Application (H.-A. Wagenknecht), p. 1985) Izvestiya Akademii Nauk SSSR: Seriya Khimicheskaya.
