Detecting Damage
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DNAHybridization an
Emil Pal ecø ek
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Miroslav
Electrochemists are developing fast and easy methods for determining nucleic
e live in an age in which molecular genetics is the new frontier. It is well known that DNA can be damaged by various physical and chemical agents (such as UV light, ionizing radiation, and photoexcited dyes) which can lead to harmful mutations, cell death, and cancer. New efforts toward diagnosing, preventing, and treating many human diseases will require a better understanding of DNA and RNA sequences. Moreover, rapid testing of various nucleotide sequences will be required for other applications, such as forensic medicine, rapid detection of biological warfare agents, and environmental testing. Developing accurate assays for DNA damage has attracted increased attention among analytical chemists. The challenge is that any new, fast assay must have sufficient sensitivity to detect one damaged nucleotide in 104–107 intact nucleotide residues, depending on the type of lesion, in microgram amounts of DNA (1–3). The development of electrochemical transducer-based devices for determining nucleotide sequences and measuring DNA damage began slowly, but recent progress is encouraging. The main advantages of these devices are their low-cost,
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F E B R U A R Y 1 , 2 0 0 1 / A N A LY T I C A L C H E M I S T R Y
75 A
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T CH3
H
O
N N
N H
C
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N O H N N
H
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G N N
N
N R
N
N
N R
A
N O
H
R
N H
FIGURE 1. Representation of Watson–Crick base pairs and electroactive groups. Circles denote sites that can be reduced at mercury electrodes; squares show sites oxidized at carbon electrodes.
simple design, small dimensions, and low power requirements. In this article, we will briefly review the present state of DNA electrochemical analysis, with emphasis on the development of the electrochemical detectors that use DNA as a recognition layer.
Analysis of nucleic acids Many techniques have been developed or adapted for analyzing nucleic acids (NAs), including electrochemistry. Electroactivity of NAs was discovered ~40 years ago (4). About 10 years later, electrochemical methods produced early evidence of DNA premelting and polymorphy of the double helix (5). When NAs interact with electrodes, they are usually strongly adsorbed. The adsorbed NAs undergo chargetransfer reactions, producing signals that can provide information about their type and concentration, changes in structure, and their interaction with various compounds. Moreover, immobilized NAs on electrode surfaces can be the basis of fast-response DNA detectors. NAs have been analyzed primarily with mercury and carbon electrodes; some work has also been reported with gold, platinum, copper, and silver solid electrodes. The potential windows for most solid electrodes is ~1 V more positive than with mercury electrodes (which operate from 0 to –2 V against the
saturated calomel electrode in neutral and alkaline pH solutions). Therefore, solid electrodes are better for studying NA oxidation, and mercury electrodes are better for investigating NA reduction. Moreover, the atomically smooth and highly reproducible surfaces of liquid mercury are very well suited for ac impedance measurements, which can provide information about DNA adsorption/desorption properties. Among NA components, only bases undergo reduction at mercury electrodes and oxidation at carbon electrodes, although sugar residues have been recently reported to be oxidized at copper electrodes using sinusoidal voltammetry (1, 6). All NA bases, and many other purines and pyrimidines, form sparingly soluble compounds with mercury, which allows them to be determined by cathodic stripping voltammetry at concentrations as low as 10–9 M (7 ). However, only free bases and purine nucleosides and nucleotides have been determined in this manner. Guanine (G) and adenine (A) residues in DNA and RNA chains are oxidized at carbon electrodes; however, their voltammetric peaks are poorly developed, providing insufficient sensitivity for DNA analysis. Recently, constant-current chronopotentiometry and sophisticated baseline correction techniques with carbon electrodes have produced well-developed oxidation peaks for DNA and RNA (Figures 1 and 2). Cytosine (C) and A residues in single-stranded (ss) DNA and RNA are reduced at mercury electrodes, producing reduction signals close to –1.4 V at neutral pH. Thymine and uracil are reduced only in nonaqueous media at highly negative potentials. In cyclic modes, G yields an anodic signal close to –0.3 V because of the oxidation of the G reduction product formed at the highly negative potentials of the background discharge.
Influence of DNA structure
The primary reduction sites of The main advantages of A and C form a part of the Watson–Crick hydrogen bonding system located in the intethese devices are their rior of the DNA duplex (Figure 1). Reduction signals of A low cost, simple design; and C are strongly influenced by DNA structure. For examsmall dimensions; andple, Figure 2 shows that, in differential pulse polarography, the reduction peak for low power requirements. (denatured) calf thymus ssDNA
76 A
A N A LY T I C A L C H E M I S T R Y / F E B R U A R Y 1 , 2 0 0 1
is absent in the parent duplex (native) calf thymus DNA polarogram, and the peak for native DNA (peak II) is almost 2 orders of magnitude smaller than the peak for denatured DNA (peak III) (5). Also, the nonFaradaic capacity signals of DNA are highly sensitive to changes in DNA structure. These signals are typically qualitatively similar to Faradaic signals and can provide information about the
2
20 nA
II
-1.5
3 µA
III -1.0
3
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2 s/V
(dE/dt)-1
1
Current
Chemically modified NAs
-1.4 -0.9 E (V)
Gox
+0.9
(b)
+1.1 Gox
III
3 µA
2 s/V
(dE/dt)-1
Current
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Current
The reduction and oxidation of natural NAs is electrochemically irreversible and occurs at highly negative or highly positive potentials. To increase sensitivity, noncovalently (DNA intercalators and groove binders) and covalently binding electroactive markers have been introduced into DNA. These markers either undergo reversible electrode reactions at lessextreme potentials or produce catalytic signals. Another group of chemically modified NAs is peptide NAs (PNAs), which have recently gained attention because of their significance for gene therapy and DNA hybridization detectors. A few examples of these DNA electroactive markers are discussed next. Ions and molecules interact with DNA in three ways: electrostatic binding along the exterior of the DNA double helix, which is generally nonspecific; groove binding, in which the bound molecule interacts directly with the edges of base pairs in the minor or major grooves of DNA; and intercalation of planar aromatic ring systems between base pairs. Electrostatic and groove binding do not usually change DNA conformation, whereas intercalation changes the torsional angles in the sugar–phosphate backbone so as to separate adjacent base pairs enough to allow insertion of the intercalator. Further changes in DNA
structure, such as unwinding or bending, can accompany the intercalation process. Most of the substances capable of interacting with DNA, particularly groove binders and intercalators, are electroactive. DNA intercalators and groove binders were used as redox indicators in the development of DNA hybridization detectors, and examples of these compounds and their electrochemical responses on binding to DNA are shown in Figure 3. Metal chelates (intercalators and DNA surface binders) have been studied by voltammetric methods (11, 12). On addition of dsDNA to electrostatic binders, such as Os(bipy)32+ (bipy = 2,2´-bipyridine), the cathodic and anodic peak potentials shifted to more negative values; intercalators (e.g., Co(phen)33+; phen = 1,10-phenanthroline) shift peaks to more positive values, suggesting stronger hydrophobic interactions (11, 12). The ability of some metal chelates to yield electrogenerated chemiluminescence was used to develop DNA hybridization detectors (12).
Current
bases’ interactions with the electrode. Because of the strong influence that DNA structure has on electrochemical signals, mercury electrodes are more suitable for studies of DNA structural transitions and local conformational changes. Experiments with mercury electrodes have identified single-strand interruptions in linear and circular DNA molecules, differences in the superhelix density of supercoiled DNAs, and superhelix density-dependent structural transitions in DNA (1, 8). Mercury electrodes are also able to discriminate between DNA and RNA. Voltammetric methods provide perhaps the most sensitive determination of trace levels of RNA in a large excess of DNA (9). However, the anodic signal for G at mercury electrodes and the oxidation peaks for A and G at carbon electrodes are less sensitive to changes in DNA structure. At a wide range of potentials, double-stranded (ds) DNA is adsorbed at the mercury electrode without any substantial unwinding. However, in a narrow potential range around –1.2 V, some changes in the structure of dsDNA, probably helix unwinding, are observed (1, 10). These changes are manifested as an increased signal, which is characteristic of ssDNA, and proceed relatively slowly; (a) tens of seconds were necessary to complete these changes in calf thymus dsDNA. No such changes are observed in covalently closed, circular DNAs, which cannot undergo unwinding for topological reasons.
I -1.5
-1.0
-1.4 -0.9 E (V)
+0.9
+1.1
FIGURE 2. Redox signals with ds- and ssDNA. (a) Experiments with 100 µg/mL of dsDNA and (b) 50 µg/mL of ssDNA. Electrochemical techniques are (1) differential pulse polarography at the static dropping mercury electrode, (2) adsorptive stripping square-wave voltammetry at the hanging drop mercury electrode, and (3) constant-current chronopotentiometric stripping analysis at the pyrolytic graphite electrode. Faradaic peak II is dsDNA, III is ssDNA; capacitive peak I is produced both by ds and ssDNA.
Current
(a)
O
CH3 O N O HN Os O N O O N R
0.4 µA
1
2
III
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–1.0 E (V) PNA NH
O
N O
O
NH
Base
DNA
Base
O Base
O
O
P
Base
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O
N O
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O Base
O O
O
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Differential capacity
(b)
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CH3 H H CH3 CH3 H3C C CH N C CH N C CH N C H O C CH O O CH3 O CH3 CH3
N
200 nA
Current
O O
G 1
N
2 Ea
–0.7
–0.5 E (V)
–0.3
FIGURE 3. Chemically modified NAs and electroactive markers of DNA. (a) Differential pulse polarographic studies with OsO4, bipy(thymine). Curve 1 represents ssDNA modified with the osmium complex; curve 2 is unmodified ssDNA. Electrode was immersed in a 5-µL solution containing 250 ng of DNA. (b) Impedance measurements at a hanging drop mercury electrode with curve 1, PNA and curve 2, DNA decamers of identical base sequences; curve 3 shows background electrolyte (0.3 M NaCl, 0.05 M sodium phosphate, pH 8.5). The colored areas in the schematic representations of PNA and DNA highlight the monomer units. (Adapted with permission from Ref. 13.) (c) Structure of echinomycin. Cyclic voltammograms of curve 1, native and curve 2, denatured DNA at a hanging drop mercury electrode and following immersion in a 10-µM echinomycin solution for 60 s.
Osmium tetroxide complexes were probably the first electroactive markers that were covalently bound to DNA. Some of these complexes, such as osmium tetroxide, 2,2´-bipy, bind preferentially to thymine residues in ssDNA, while others, such as osmium tetroxide, phen, bind both ds- and ssDNA. DNA adducts with these complexes produce reversible signals between –0.2 and –0.7 V at both carbon and mercury electrodes, and a high catalytic signal at ~ –1.2 V at mercury electrodes (Figure 3a). With the catalytic signal, sensitivity is 2 orders of magnitude better than the signal of unmodified ssDNA. Other electroactive markers were later coupled to NAs, primarily for use in biosensing. In PNAs, the entire sugar– phosphate backbone is replaced by N-(2-aminoethyl) glycine units (Figure 3b). This drastic structural change results in NA mimetics with higher binding affinity to complementary DNA and RNA sequences than unmodified oligonucleotides. This is surprising because even minor structural changes in oligonucleotides, such as the replacement of an oxygen atom by a methyl group (methyl phosphonates) or by sulfur (phosphorothioates), lower binding affinity. PNA is achiral and has an electrically neutral backbone. It produces electrochemical responses at carbon and mercury electrodes similar to DNA, and the reduction of A, C, and G occurs at mercury electrodes. Peak potentials of ssPNA are shifted to more negative values than ssDNA. Differences in the structures of PNA and DNA are mani-
fested by the different adsorption behavior of these two compounds as detected by ac impedance (Figure 3b) and chronopotentiometric measurements at mercury and carbon electrodes. At higher surface concentrations, PNA molecules associate at the mercury surface. Prolonged exposure to highly negative potentials does not desorb PNA molecules; although, under these conditions, almost all DNA molecules are removed from the surface. PNA adsorption increases with decreasing salt concentration, whereas DNA adsorption decreases under the same conditions (13). For DNA hybridization detectors, PNAs may serve as better probes than DNA.
DNA conductivity
which has been demonstrated by many authors using different approaches.
DNA hybridization A biodetector or a biosensor consists of a selective biological recognition element associated with a transducer, which translates the recognition event into a physically measurable value (20). In an electrochemical DNA hybridization detector, a short ssODN (a DNA probe usually 15–20 nucleotides long) is usually immobilized on a transducer (electrode) to create a DNA recognition element. The probe-modified electrode is then immersed into a solution of target DNA. When the target DNA contains a sequence that exactly matches that of the probe DNA, a hybrid duplex is formed at the electrode surface. In the absence of a complementarity between the probe and target, no duplex is formed. (Partial complementary binding of these DNAs may result in a weak interaction that is more difficult to recognize.) Hybrid formation is then translated into an electrical, analytically useful signal. The two most important abilities of a hybridization detector are forming a DNA recognition layer and generating the electrical signal following the hybridization event. The choice of electrode material and the method of DNA immobilization play critical roles. To immobilize probe DNA, both covalent and noncovalent binding have been used. Adsorption forces were used to bind DNA to carbon and mercury surfaces. Electrostatic binding of DNA to positively charged carbon electrodes is sufficiently strong, leaving the bases accessible to interact with target DNA. Even stronger binding of DNA to mercury surfaces occurs because of hydrophobic interactions of bases with the surface, which prevents any efficient DNA hybridization. Various kinds of covalent binding of DNA to carbon, indium–tin oxide, gold, and mercury surfaces have also been used (16, 21–23). Random covalent binding of DNA to electrode surfaces involving chemical modification of bases decreases the specificity of the recognition layer and therefore is not recommended. Significantly better results can be obtained by immobilizing the probe via one end of the DNA molecule, such as in thiolated ODNs. Generally, a covalently bound probe is better because it provides more opportunities for easily removing the nonspecifically bound molecules. So far, DNA hybridization probe immobilization onto various surfaces has not been systematically investigated. It appears, however, that carbon and gold surfaces
The two most important abilities of a hybridiza tion detector are forming a DNA recognition layer and generating the elec trical signal following hy
Whether DNA can mediate charge transfer has been discussed for several decades. Recent fluorescence quenching experiments have renewed the debate (14). It was shown that, when nucleobase radical ions form within the DNA duplex, a long-range charge transfer could occur, which required properly spaced G residues (15, 16). Attaching a thiol group to one end of an oligodeoxynucleotide (ODN) made it possible to form self-assembled monolayers of ss- or dsDNAs on gold surfaces. Recently, daunomycin covalently bound to the 2-amino group of G in 15-bp thiol-modified duplexes was observed to undergo efficient reduction regardless of its position in the duplex (17 ). The presence of a C–A mismatch between daunomycin and the electrode surface completely abolishes the electroreduction of daunomycin. These results indicate that electron transfer is blocked by perturbations in base stacks induced by base mistakes, and they might support the concept of long-range charge transfer within the -stack of the DNA duplex. It was also recently found that a disordered chromosomal dsDNA film was not able to “wire” to the enzyme soybean peroxidase (which reduces H2O2 to water). It was suggested that randomly oriented dsDNA does not conduct electrons or holes (18). On the other hand, dsDNA, an ordered solid 12-mer film aligned to the gold electrode via the end thiol, displayed semiconductor characteristics. Measurements of electrical transport through individual poly(dG).poly(dC) molecules (DNA duplex composed of polyguanylic and polycytidylic homopolynucleotide chains), which were connected to two metal nanoelectrodes, indicated semiconductor behavior for these molecules (19). Substantial progress in DNA conductivity has been achieved, but many questions still remain to be answered. One of the most exciting discoveries is the change in the electrical properties of dsDNA upon introduction of a base mismatch,
F E B R U A R Y 1 , 2 0 0 1 / A N A LY T I C A L C H E M I S T R Y
79 A
are well suited as electrode materials, with the latter surface requiring covalent attachment of DNA. Redox indicators. Electrochemists have used redox indicators to differentiate between ds- and ssDNA. Two unique intercalators—threading intercalators and bis-intercalators—are particularly interesting as redox indicators. The threading intercalators have substituents on opposite sides of the intercalating aromatic ring system and must thread one of the substituents between the base pairs at the intercalation site. Threading in-
tercalators with large side chains force the helix to open wide and significantly distorts the double helix structure, making the kinetics of intercalation much slower than with simple intercalators. On the other hand, the large side chain intercalators can have high DNA binding constants, which indicate that once the substituent slides between the base pairs, DNA assumes a conformation with a very favorable free energy of complex formation. Bis-intercalators have two intercalating rings covalently linked with connecting chains of various lengths. There are also multiintercalators containing three or more intercalation rings, which were synthesized because, as potential drugs, their high DNA binding constants are expected to enhance their (a) therapeutic activity. Electrochemical studies of the synthetImmobilized probe ic threading intercalator ferroReporter probe with Target cenyl naphthalene diimide and ferrocene marker DNA the naturally occurring bis-intercalator echinomycin, an antibiotic and antitumor agent, showed that both compounds bind to dsDNA more tightly than usual intercalators (Figure 3c) but have low affinities for ssDNAs. Using ferrocenyl napthalene diimide, the yeast choline transport gene was re(b) cently detected (24). In principle, target DNA Electrode can be end-labeled covalently H2O with a redox indicator that produces a signal when a probe binds. This is rather inconvenient, because each target DNA has to be labeled, and the end-label on a long Peroxidase DNA segment may not get coupled to e– oxidized or reduced at an target DNA H2O2 electrode covered with DNA. Another method relies on Conducting polymer an end-labeled ODN that is complementary to the target FIGURE 4. Hybridization systems using reporter probe or catalytic electroreduction. (reporter probe), which is bound so that the label is next (a) An ODN bearing an electroactive marker that complements a target DNA sequence near the site of attachment. Sigto the hybridized probe nal of the reporter probe marker (ferrocene) is measured. (Adapted with permission from Ref. 25.) (b) A probe is covalently bound to a conductive polymer coating the electrode surface; target DNA is coupled to thermostable soybean (Figure 4a). Ihara et al. used peroxidase. Upon hybridization, an electrocatalytic current created by hydrogen peroxide reduction is observed. (Adaptthis approach with a fered from Ref. 18.) rocene-modified ODN and a probe DNA connected to a gold electrode via five successive phosphorothioate units
on the 5´ terminus (25). Indicator-free systems. The electroactivity of NAs can be used to detect DNA hybridization. a Probe Reporter probe Because of the large differences in the electrochemical responses of ds- and ssDNAs at mercuDifferences in properties Target DNA ry electrodes, these electrodes should be highly of ss versus dsDNA suitable for detecting DNA hybridization. Our preliminary results support this. The DNA reb Enzyme/HRP sponses at solid electrodes depend less on DNA structure, making these electrodes less suitable for differentiating duplexes from ssDNA. Redox indicator G G GG Any electrode can, however, distinguish the c G probe and target DNAs, if their base contents are FIGURE 5. Detection of hybridization using reporter probes or sufficiently different. The strategy is to replace catalytic reduction. electroactive G residues in the probe sequences with electroinactive hypoxanthines, which still (a) Reporter probe binds to a complementary sequence in target DNA bind to C bases. Hybridization is detected near the electrode surface and (b) reporter enzyme attached to target DNA before hybridization. (c) DNA probe lacking any G bases is used. through the target DNA G signal (Figure 5). The presence of G in target DNA is detected after the hybridization This approach has been used to detect DNA hyusing G oxidation signals on indium–tin oxide or carbon electrodes. bridization at indium–tin oxide and carbon electrodes (22, 26). However, substituting hypoxanthines in the probe can decrease the stability of the duplex and the specificity of the hybridization. Another approach is to use ac impedance and, particularly, ture for a given polymer. This signature is very sensitive to the impedance spectroscopy for probing the interfacial properties environment, and different functionalities can be inserted into (e.g., capacitance and electron-transfer resistance) of modified the polymer. Conducting polymers, such as copolymers functionalized electrodes. The large differences in capacitance measurements arising from the interfacial properties of ss- and dsDNAs have with osmium complexes, polyazines, polyanilines, polypyrroles, been shown to recognize DNA hybridization (1, 27, 28). Re- or polythiophenes, can be used for blocking and interfacing the cently, a three-component ODN system on a gold electrode transducer, modulating DNA interactions at surfaces, and genwas used to detect specific DNA sequences by Faradaic imped- erating signals (30). For example, conducting polypyrrole funcance spectroscopy (29). Conductivity of the perfectly matched tionalized with a bulky ODN was electroactive in aqueous duplex DNA combined with lack of conductivity in duplexes media, and after interacting with the complementary ODN, it containing a base mismatch may be of great use in future DNA showed a change in the voltammetric signature (31). Another hybridization detectors. More information is needed about the example used NAs adsorbed on polypyrrole-coated carbon behavior of ssDNA at electrodes. electrodes to detect ODNs in flowing streams (32). Doping the NA probes within electropolymerized polypyrrole films and Blocking and interfacing the transducer monitoring the current changes due to the hybridization apSo far, in most experimental models, ODNs have been used as pear to be a promising label-free biosensing strategy (33). target DNAs. However, with real DNA samples, nonspecific inTwo-component films have been prepared containing a teractions at the electrode surface, such as those arising from thiol-derived ssDNA probe and a diluent thiol, mercaptohexalong DNA chains or DNA impurities, can obscure the hybrid- nol, which prevents nonspecific adsorption of the ssDNA (34). ization signals. To prevent this, researchers are developing more Two-color surface plasmon resonance measured the dielectric efficient means of interfacing between the DNA system and the constant and thickness of this film. The amount of DNA tethelectrode surface. Thiols and conducting polymers have been ered to the surface was quantified, and the kinetics of hyused for this purpose. bridization and thermally induced dehybridization were deterConducting polymers are synthesized chemically or electro- mined—all indicating a high efficiency for hybridization. polymerized as thin films onto an electrode. Experimentally obThe electrode processes for detecting the hybridization events tained conductivities of conjugated polymers are several orders described so far involve one or few electrons and are, therefore, of magnitude lower than that of metals, but sufficiently high to inherently low-yield reactions. An alternative strategy would be consider these macromolecules as molecular wires. Reversible to collect as many electrons as possible, facilitated by some catredox processes in these polymers can be potential-controlled, alytic processes. Thorp demonstrated this approach by using a and cyclic voltammetry can provide an electrochemical signa- soluble mediator that moved close to G bases present only in
F E B R U A R Y 1 , 2 0 0 1 / A N A LY T I C A L C H E M I S T R Y
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target DNA and then shuttled electrons quickly to a polymermodified indium–tin oxide electrode (22). The electrode potential was held at a sufficiently positive value to oxidize the reduced form of the mediator [Ru(bipy)3]2+. The oxidized form of the mediator then removed electrons from G residues, generating reduced [Ru(bipy)3]3+ and completing the catalytic cycle. Under favorable conditions, ~100 electrons per hybridized G could be collected, affording a high sensitivity.
duplex and a single base mismatch duplex; at room temperature, such discrimination is not possible. What makes this result so important is that previous papers have demonstrated either improved sensitivity or specificity, but Caruana and Heller offer, for the first time, a method that demonstrates both properties. As such, their work is a significant step toward a practical electrochemical hybridization detector. All hybridization detectors developed to date require probe and target ssDNAs. Interestingly, dsDNA carrying a homopurine/homopyrimidine segment can capture the homologous homopyrimidine ODN, and this observation could open the way for developing electrochemical procedures for affinity capture of triplex-forming ODNs. The triplex strategy, particularly in combination with PNAs, is capable of invading the DNA duplex and forming highly stable complexes.
Detecting point mutations Many diseases are connected with a single base or point mutation. Detecting such a small change in the DNA duplex is difficult and, therefore, requires highly specific methods. Recently, we detected single base mismatches in different DNA sequences, including a mutation hot spot in the p53 gene, by using PNA instead of DNA (35, 36). Caruana and Heller have recently shown that a single base mismatch in an 18-mer ODN can be detected with a redox polymer-coated microelectrode in a system that uses peroxidaselabeled target DNA (Figure 4b) (37). Using the thermostable soybean peroxidase enzyme, they obtained excellent discrimination at elevated temperatures between a perfectly matched
scDNA
Ionizing radiation,
Strand break
radicals, nucleases
Open circular (nicked) DNA
Gox
Mercury electrode
Detecting DNA damage Two approaches are used to detect damaged DNA. In the first, DNA is hydrolyzed, and damaged entities are separated by HPLC, GC, or CE. Lesions are detected on-line by various methods, such as MS, amperometry, fluorescence, and radioactivity. The other approach leaves DNA intact, and lesions are
Gox
3
Carbon electrode
Mercury electrode
Carbon electrode
FIGURE 6. Detection of DNA damage with scDNA-modified electrodes. AC voltammetry at a mercury electrode shows the appearance of a new peak labeled 3. The peak arises because the interruption of the sugar phosphate backbone makes bases accessible in the vicinity of the break.
measured by immunoassays, sedimentation, or gel electrophoresis to quantify the number of strand breaks. Damage to bases can be transformed into strand breaks due to the nicking activity of DNA repair enzymes or by chemical treatment. All these techniques are very useful, but none are fast enough to provide information in seconds or minutes. The high sensitivity of polarographic methods for detecting single-strand breaks (ssbs) in linear DNAs has been known for decades (1). Recently, we developed a method for determining one ssb among more than 2 ⫻ 105 intact phosphodiesteric bonds in nanogram amounts of DNA (38). Circular covalently closed supercoiled (sc) DNA is immobilized at a hanging mercury drop electrode (HMDE) (Figure 6). If there is a strand break, a new ac voltammetric capacitance peak (or a constant-current chronopotentiometric Faradaic peak) is observed. These signals are caused by bases at the strand break interacting with the electrode. In these experiments, the DNA is immobilized on the electrode by a short incubation of HMDE in about 5 µL of DNA solution, followed by washing and transfer of the modified electrode into an electrochemical cell containing blank background electrolyte. Using this approach, either DNA damaged in solution can be immobilized at the surface or the electrode can be modified with intact scDNA, which is used to detect DNA-damaging agents in the medium. Recently, both detector systems have been used to detect hydroxyl radicals in laboratory-prepared solutions and the latter system was used to detect DNA damaging agents in natural and industrial waters. To avoid using liquid mercury in the field, a mercury film carbon electrode can be used instead. This detector responds in seconds and, thus, is much faster than popular electrophoretic methods. Recent advances in DNA hybridization and damage detectors are encouraging. It is only a question of time when these types of electrodes will become standard elements of microfabricated devices for integrated NA analysis (39). This work was supported by grants 204/97/K084 and 204/98/P091 from the Grant Agency of the Czech Republic.
Emil Palec˘ek is professor of molecular biology and head of the Laboratory of Biophysical Chemistry and Molecular Oncology at the Institute of Biophysics (Czech Republic). His research interests include chemical reactivity and structure of nucleic acids, electrochemical analysis of biomolecules, and DNA–protein interactions in oncology. Miroslav Fojta is also at the Institute of Biophysics. His research interests include electrochemical analysis of nucleic acids, detection of DNA damage, and DNA interactions with tumor suppressor protein p53. Address correspondence to Palec˘ek at the Institute of Biophysics, Academy of Sciences of the Czech Republic, Královopolská 135, 612 65 Brno, Czech Republic (4205-746-241;
[email protected]).
References (1)
Palec˘ek, E. In Topics in Bioelectrochemistry and Bioenergetics; Milazzo, G.,
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