Fig. 8. Electrochemical detection of a single CA mismatch using both (A) cyclic voltammetry and (B) chronocoulometry.

Fig. 8. Electrochemical detection of a single CA mismatch using both (A) cyclic voltammetry and (B) chronocoulometry.

is collected and the volume is reduced and purified by HPLC (analogously to DNA-5'-NH2) and dried in vacuo. Next, the DNA-5'-SS product is resuspended in 200 fil of PBS. Dithiothreitol (DTT, 3 mg) is then added and the reduction reaction proceeds at ambient temperature for 40 min to form the final product, DNA-5'-SH. This is gel filtered on an NAP-5 column (Sephadex G-25, DNA grade) in PBS and then HPLC purified and dried in vacuo.

The thiol-modified single strand (DNA-5'-SH) can be tested for the presence of free thiol via HPLC by Ellman's test as follows. A small aliquot 100 pi) of the DNA-5'-SH HPLC fraction is reinjected onto the HPLC for an analytical run followed by a second 100-/il aliquot to which 1 /il of 10 mM dithionitrobenzoic acid (DTNB; Ellman's reagent) had been added. Free thiol is monitored by a shift in the DNA-5'-SH chromatogram peak as well as new peaks in the UV-Vis spectrum at 330 and 410 nm.

The single-stranded oligonucleotide complementary to the thiol-modified strand is synthesized (trityl-off) at a l-/imol scale on a DNA synthesizer using standard solid-phase phosphoramidite chemistry (1000-A CPG). The CPG-bound DNA is then transferred to a 1,5-ml Eppendorf tube and mixed with 1 ml of concentrated NH4OH and incubated at 55° for 8 hr to cleave the oligonucleotide from the CPG resin and deprotect all the bases. This product is then cooled, decanted, and evaporated to dryness in vacuo. This product is purified by reversed-phase HPLC.

The two purified, complementary single-stranded oligonucleotides are then quantitated and hybridized to make thiol-modified duplexes. Oligonucleotide stock solutions are prepared and quantitated by UV-Vis spectroscopy [/.max = 260 nm, e (M ~1 cm"1)]. The extinction coefficient of single strands is calculated by the sum of the extinction coefficients of the individual bases: e(dA) = 15,400, e(dG) = 11,500, e(dC) = 7400, e(dT) = 8700. Duplexes are formed by combining equimolar amounts of each strand in PBS for a final solution of 100 11M duplex. This solution is degassed and blanketed with argon, heated to 90° for 5 min, and then cooled slowly to room temperature (2 hr). Just before deposition on the clean gold electrode, 100 mM MgCl2 is added to each sample.

Preparation of Electrode Surface. The gold electrodes are prepared by standard procedures. They are polished with 0.05-/xm alumina, sonicated in distilled H20 for ~20 min, electrochemically etched in 1M H2S04, and rinsed well with distilled H20. The electrodes are then inverted and a 10-/il drop of the thiol-modified DNA duplex solution is deposited onto each electrode surface. The electrodes are kept in a moist environment at room temperature during the assembly process. To ensure maximum density of the monolayer, self-assembly is usually allowed to proceed overnight, but assembly does proceed much faster if the thiol and gold surface are perfectly clean. After assembly, the complementary DNA strand can be removed and replaced with test strands by in situ hybridization as follows. The DNA electrode is immersed in 90° PBS for 5 min and then rinsed thoroughly in PBS. Next, the electrode is immersed in a solution of 100 pM test strand oligonucleotide in

PBS with 100 mM MgCl2 at 90° and allowed to cool to room temperature. Alternatively, if test strand is limited in quantity, a drop of the test strand in PBS with 100 mM MgCl2 can be placed on the hot electrode surface and allowed to cool. During the cooling process, it is important to prevent evaporation of the solution, so drops of PBS should be placed on the surface as needed.

Electrochemical Assays for Single-Base Mismatch Detection at DNA-Modified Surfaces. Cyclic voltammetry (CV) is carried out in a two-compartment cell filled with PBS that is degassed and blanketed with argon. The DNA-modified gold working electrode and the platinum wire auxiliary electrode are separated from the saturated calomel reference electrode (SCE) by a modified Luggin capillary. Before electrochemical analysis, excess DNA is rinsed away with PBS and monolayer coverage is qualitatively checked with 2 mM Fe(CN)63_ in PBS by scanning the potential from 0 to 600 mV and back at 100 mV/sec. If a CV signal is not observed, it is interpreted that the monolayer is so dense that Fe(CN)g3~ cannot diffuse to the gold surface and participate in redox chemistry. Thus in the following electrochemical studies of DNA intercalators, it is assumed that any CV signal observed is the result of charge transport through the DNA monolayer. Monolayers can be extensively characterized by AFM, ellipsometry, and radiolabeling of the duplexes, which is described elsewhere.47,48

If the electrode surface is sufficiently covered, a catalyst such as MB is added to the solution for a final concentration of 0.5 ¡lM. Another catalyst can be used, but it must satisfy these requirements: it must bind to DNA by intercalation, it must not bind either too tightly or too loosely (the catalyst must be able to access the DNA base stack as well as the ferricyanide in solution), and its reduction potential must be about —200 to —600 versus SCE. This protocol assumes MB is used as the catalyst; if another intercalator is chosen, the electrochemistry conditions will need to be optimized. After addition of the catalyst and degassing with argon, the potential is scanned from 0 to —600 mV (vs. SCE) at 100 mV/sec. If the duplexes on the surface are well packed and contain no mismatches or other tc-stack-disrupting lesions, a large catalytic wave is observed at about —350 mV. If there are mismatches, the background will start to rise at about —600 mV, but there will be no distinct peak (Fig. 8).

A more sensitive way to check for mismatches is to carry out chronocoulometry. The electrode potential is stepped to —350 mV versus SCE and allowed to integrate for 5 sec. If there are no base stack perturbations, the charge should steadily increase over the course of the experiment; the maximum amount of charge accumulated depends on the sequence, but typically the charge will reach 16 to 25 /iC. If there is a mismatch, only 1 to 6 /iC will accumulate, depending on the extent of perturbation and the sequence (Fig. 8).

0 0

Post a comment