ANALYTICA CHIMICA ACTA

Analytica Chimica Acta 342 (1997) l-12 ELSEVIER

Supercoiled DNA-modified mercury electrode: A highly sensitive tool for the detection of DNA damage’

Miroslav Foj ta, Emil PaleCek*

Institute of Biophysics of the Academy of Sciences of the Czech Republic, Krdovopolska 135, 612 65 Bmo, Czech Republic

Received 27 August 1996; received in revised form 11 November 1996; accepted 13 November 1996

Abstract

A fast method for the detection of small damage in DNA is presented. The method is based on the appearance of an a.c. voltammetric peak (peak 3) resulting from the introduction of a single-strand scission into the covalently closed circular (supercoiled) DNA molecule. Peak 3 is absent in an intact supercoiled DNA. DNA is immobilized by short incubation of a hanging mercury drop electrode in 5 ~1 of the DNA solution followed by washing and transfer of the DNA-modified electrode in a voltammetric cell containing blank background electrolyte. This makes it possible to determine one strand scission in <2% of the DNA molecules or among >2.5 x lop5 phosphodiesteric bonds. The amount of DNA required for the analysis is below 10 ng. The DNA damage can be studied either in solution or the electrode modified with intact supercoiled DNA can be used as a sensor to detect the DNA damaging agent (e.g. hydroxyl radical) in the environment. In the latter case the response can be obtained in seconds. The method is faster than the currently applied gel electrophoresis.

Kq~words: Supercoiled DNA; DNA damage; Voltammetry

1. Introduction

Damage to DNA in eukaryotic cells may result in

serious disturbances of the cell life including apopto- sis or malignant cell transformation [l]. A number of physical and chemical agents capable to damage DNA in various ways exist in the environment. Interruption of the DNA sugar-phosphate backbone can be sensi- tively detected in supercoiled DNA molecules by agarose gel electrophoresis [2,3]. A disadvantage of this method is that the analysis takes at least several

*Corresponding author. Tel.: (4205) 415 17177; fax: (4205) 412

11293; e-mail: palecek@zoncna.ibp.cz.

‘In memory of Professor Julius Marmur.

hours and that quantitation of the ethidium bromide stained gels is rather difficult. Compared to the gel electrophoresis electrochemical methods are faster and quantitation of data is easy.

Using adsorptive stripping voltammetry (AdSV) DNAs can be analyzed at DNA concentrations below 1 ug ml-’ but the volumes of the DNA solution necessary for the analysis are much larger (at least l-2 ml) [4,5] than those used in gel electrophoresis (usually 10-20 ~1). Recently we have shown [4,6-121 that a DNA-modified hanging mercury drop electrode (HMDE) can be prepared using 4-10 ~1 volumes of a DNA solution. The electrode is then transferred into the electrolytic cell containing an empty background electrolyte where the voltammetric measurements are

0003-2670/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SOOO3-2670(96)00551-X

2 M. Fojta, E. Palefek/Analytica Chimica Acta 342 (1997) I-12

performed. This procedure is called adsorptive trans- fer stripping voltammetry (AdTSV).

We have shown that using AdTS cyclic voltamme- try (CV) subnanogram amounts of DNA can easily be analyzed and that the heights of the DNA cathodic peak CA (due to electroreduction of cytosine and adenine residues) and anodic peak G (due to guanine) at a given DNA concentration differ for supercoiled (SC), linear (lin) and denatured (den) DNAs [ 111. The difference between the peak heights of double- stranded linear and single-stranded denatured DNAs is sufficiently large that it can be used to study the DNA helix-coil transition. Here we show that a.c. voltammetry, at weakly alkaline pH, represents a better system suitable for the detection of the DNA damage by physical and chemical agents. Single- strand scissions of supercoiled DNA result in an appearance of a specific (non-faradaic) peak not pro- duced by intact scDNA. Using this system we have been able to detect one single-strand scission per DNA molecule induced by y-radiation or by ‘OH radicals (generated by iron(II)/EDTA complex) in less than 2% of the DNA molecules. The DNA strand scission has been measured either in DNA solution or using scDNA-modified electrode as a sensor.

2. Experimental

2.1. Material

Plasmid DNAs [pBSK(-), pUC19, pBR322] [2] were isolated from chloramphenicol-amplified HB 101 Escherichia coli cells using (1) boiling method (including phenol extraction and LiCl precipitation) [2], (2) alkaline Birnboim method [2], or (3) lyso- zyme/SDS/EDTA lysis [ 131. All procedures included l-2 isopycnic centrifugations in CsCl/ethidium bro- mide gradient followed by repeated extraction with cold butan-l-01, extensive dialysis against 10 mM Tris, 1 mM EDTA buffer (TE buffer), pH 7.9 and ethanol precipitation [2]. Plasmid pAT32 DNA, iso- lated by Birnboim procedure followed by QIAGEN column chromatography [ 141 and CsCl-gradient cen- trifugation was a gift of Dr. Karel Nejedly (Bmo). Calf thymus DNA was isolated as described before [9].

Linear plasmid DNAs were prepared by EcoRI or Hind111 (Fermentas) cleavage of scDNA [2]. To pre-

pare open circular (oc) DNA, scDNA was incubated with 0.02 U ml-’ of deoxyribonuclease I (Sigma) in 6 mM MgCl*, 2 mM CaC12, 40 mM Tris-HCl pH 7.4 at 0°C for 90 min (conditions under which one single- strand scission per DNA molecule is formed were pretested). Lin or ocDNA were deproteinized by extraction with a mixture of phenol, chloroform and isoamylalcohol (25 : 24 : l), twice extracted with chloroform and precipitated with ethanol [2]. Dena- turation of lin or ocDNA was performed by heating of the sample (in 10 mM TE) for 5 min at 96°C followed by rapid cooling on ice bath. Fig. 1 shows the relations between the different plasmid DNA forms and gel electrophoresis of them. Other chemicals were of analytical grade.

2.2. Methods

2.2.1. Voltammetric measurements Alternating current voltammetric measurements

were performed with an EG&G PAR 174A Polaro- graphic Analyzer connected to 174150 AC Polaro- graphic Interface, 5208 Two Phase Lock-in Amplifier and a Philips PM 8134 X, Y1 Y2 Recorder. Following parameters were chosen: frequency 230 Hz, peak-to-peak amplitude 10 mV, scan rate 20 mV s-l; both phase in and phase out components were recorded (preferably the phase in component- due to better resolution of the DNA signals). For other parameters see Section 3 and the figures. Linear sweep voltammetry was performed with a PAR 362 Scanning Potentiostat (scan rate 0.2 V s-l). A Metrohm 647 VA-Stand electrode in HMDE mode controlled by 646 VA-Processor was used. All mea- surements were performed with saturated calomel reference electrode (SCE) and a platinum wire counter electrode. 0.3 M NaCl, 50 mM sodium phosphate, pH 8.5 was used as a background electrolyte. The way in which heights of DNA peaks 1 and 3 (It and Is, respectively) were measured is shown in Fig. 2(c).

2.2.2. Preparation of nucleic acid-modified electrodes

DNA was adsorbed at the electrode surface from 5 pl drops of solution containing 0.2 M NaCl and 10 mM Tris-HCl pH 7.4, if not stated otherwise. The DNA-modified electrode was washed twice by distilled water and by background electrolyte solution

M. Fojta, E. PaleEek/Analytica Chimica Acta 342 (1997) 1-12 3

96 “C ,~~~~-~-~~~~~-~~-~__.~

b

QQ J+ den --- ____ _____ ______ ____ /

C

Fig. 1. (a) Scheme of the relations between SC, DC, lin and denDNA. Both strands of scDNA are covalently closed thus preventing DNA

unwinding (including denaturation at 96°C). As results of the formation of a single-strand break (ssb, induced e.g. by ionizing radiation) or of

a double-strand break (dsb, produced e.g. by a restrictase), oc or hnDNAs are formed, respectively. The latter DNA forms (containing breaks

in their strands) can be denatured, (b) ethidium bromide-stained agarose gel (1%) electrophoresis of SC (track 1). oc (track 2) and tin (track 3) pBSK(-) DNA (the same SC and 1inDNA samples were used in Fig. 2), 400 ng per track, (c) ethidium bromide-stained agarose gel electrophoresis of pUC19 scDNA sample (tracks 1, 5); the same DNA irradiated with a dose of 16 Gy (tracks 2, 3) and 150 Gy (track 4). Track

3, the sample as in track 2 exposed to 96°C for 5 min after y-irradiation (see Fig. 6); in tracks 14, 300 ng of DNA; in track 5, 3 ug of DNA

were loaded.

and transferred into deareated blank background electrolyte, which was then bubbled with argon for 90 s. The initial potential (Ei) was applied at the electrode for 15 s (quiescent period) prior to the voltage scan.

2.2.3. Gel electrophoresis Samples of DNA (300-500 ng) were loaded on 1%

agarose gel containing 40 mM Tris-acetate, 1 mM

EDTA, pH 7.8. After separation (voltage density usually 34 V cm-‘) the gels were stained with 1 ug ml-’ ethidium bromide, and photographed.

2.2.4. Irradiation of DNA Samples of scDNA (100 ug ml-‘) were y-irradiated

by a Chisostat “Co source in Eppendorf tubes con- taining 20 pl of the DNA solution in 0.2 M NaCl, 10 mM TE buffer pH 7.4, at room temperature.

4 M. Fojta, E. Pale&?k/Analytica Chimica Acta 342 (1997) 1-12

i 1 0.5 pA dkJ I3 : I i : i : !J Ii

1” i . . . . . . . . . ..-.. .._. _ ..,.,.._......_, _,,

C I

1

-I .5 -1 .o -0.5

f 1

I

-1.4 -1.0

potential (V vs. SCE)

Fig. 2. Adsorptive transfer stripping ax. voltammograms yielded

by supercoiled (a,d); linear double-stranded (b,e); and thermally

denatured (c,f) pBSK(-) plasmid DNA. (a)-(c) full coverage of the electrode; r,=120 s, DNA concentration: SC, 15Opg ml-‘; lin,

8Opg 1-I; den, 45 pg ml-‘; (d)-(f) partial coverage of the

electrode; rA=120 s, DNA concentration: SC, 5 pg ml-‘; lin and

den, 4 pg ml-‘. DNA was adsorbed at the electrode from a 5 pl

drop of solution containing 0.2 M NaCl, 10 mM Tri-HCl pH 7.35

for rA= 120 s. Alternating current voitarnmetry, in phase, frequency

230 Hz, peak-to-peak amplitude 10 mV, scan rate 20mV s-‘,

initial potential -0.1 V. Dashed line in (a), background electrolyte;

in (c), the way of peak height measurement is shown.

2.2.5. Treatment of DNA with Fe/EDTA reagent Supercoiled DNA was treated with the reagent (both

in solution and at the electrode) in 0.3 M NaCl, 50 mM sodium phosphate buffer pH 8.5. The reaction mixture contained FeS04, EDTA and sodium ascorbate at the ratio 1 : 2 : 20; the H202 concentration was kept con- stant at 0.03% (v/v) [15] (for example “50 PM Fe/ EDTA reagent” contained 50 pM FeS04, 100 pM EDTA, 1 r&l sodium ascorbate and 0.03% H202).

Treatment in solution. DNA at a concentration of 150 pg ml-’ was incubated with the reaction mixture for 60 s prior to adsorption at the electrode (for

tA=60 s from the same solution without any further

treatment). Cleavage at the electrode. HMDE fully covered

with scDNA was incubated in the [Fe(EDTA)]*- reagent for 1 min; the electrode was then washed and transferred into blank background electrolyte. For gel electrophoresis, reaction was stopped by an addition of 100 mM thiourea and the DNA was ethanol precipitated.

3. Results and discussion

3.1. a.~. voltammetric responses of supercoiled (SC), open circular (oc), linear (lin) and denatured (den) DNA at full electrode coverage

HMDE was immersed into a 5 ~1 drop of the pBSK(-) DNA solution for the accumulation time tA=l20 s. The DNA concentration was adjusted to secure full coverage of the electrode under the given conditions (SC 150 pg ml-*, lin or oc 80 pg ml-‘, den 40 pg ml-’ in 0.2 M NaCl, 10 mM Tris-HCl buffer pH 7.4). The electrode was washed and transferred into the electrolytic cell containing a blank back- ground electrolyte to perform the a.c. voltammetric measurement. Supercoiled DNA yielded peak 1 (at E,=-1.17 V, Fig. 2(a), Table 1) and small inflection 2 around - 1.3 V. In addition to these signals linear- ized (lin) DNA (Fig. 2(b), Table l), as well as open (nicked) circular (oc) DNA produced peak 3 at more negative potentials (EP=- 1.43 V). Peak 1 of ocDNA

Table 1 Potentials and heights of AdTS a.c. voltammetric peaks of SC, oc,

lin and den pBSK(-)DNA

SC oc

Phase in

Peak 1 Ep (V) -1.17 -1.19

Ip (nA) 182 205 Peak 3 Ep (V) - -1.43

1, (nA) - 190 Phase out

Peak 1 Ep (V) -1.17 -1.20

1, @A) 12.0 14.5

Peak 3 Ep (V) - -1.43

1, @A) - 13.5

For experimental conditions, see Fig. 2.

Lin den

-1.19 -1.19

293 289 -1.43 -1.43

120 470

-1.18 -1.19

18.3 19.5 -1.43 -1.42

8.5 22.3

M. Fojta, E. PaleEek/Analytica Chimica Acta 342 (1997) l-12 5

was about 12% higher than that of scDNA, and that of IinDNA was higher by about 60%. Compared to linDNA, thermally denatured DNA produced much higher peak 3 (E,=-1.43 V); peak 1 (&=- 1.20 V) of denDNA was about the same as that of 1inDNA (Fig. 2(c), Table 1). All these signals were observed both on phase in and phase out component of the current response (Table 1); the phase in peaks gave current values by two orders of magnitude lower but they were better resolved than the peaks yielded by the phase out component of the current. Due to this fact, the phase in curves were measured in this paper.

Similar results were obtained with other plasmid preparations. Freshly prepared supercoiled plasmid DNAs purified by at least two CsCl-gradient centri- fugations or by one gradient centrifugation followed by a QIAGEN chromatography (showing strong scDNA band but no ocDNA band on the agarose gel, Fig. l(b)) typically produced no peak 3. [With samples purified only by one centrifugation a small but significant peak 3 was usually observed; this peak was probably due to the presence of ocDNA (detect- able on an overloaded agarose gel, Fig. l(c), track 5) or some short DNA chains (weakly bound to scDNA) contaminating the sample of scDNA.1 Heights of peak 1 yielded by SC and ocDNA were always smaller than those of lin and denDNA. The differences in the peak heights obtained with different DNA samples [pUC 19, pBR322, pAT32, pBSK(-)I (at full electrode coverage) did not exceed 20%. Differences in the Er, values were below 10 mV.

3.2. Dependence on DNA concentration

At partial electrode coverage, peaks of SC, lin and den pBSK(-) DNAs (Fig. 2(d)-(f)) did not qualita- tively differ from those obtained at a full electrode coverage (Fig. 2(a)-(c)) with an exception of the inflection 2 which was not observed at lower concen- trations of DNA. At tA= 120 s (without stirring) peak 3 showed linear dependence on concentration of denDNA in the range from 1 to almost 15 pg ml-’ (Fig. 3(b)). At about 20 pg ml-’ the height of peak 3 of denDNA reached its limiting value (about 500 nA) which was independent of the DNA concentration up to the highest concentration measured (40 pg ml-‘). In stirred solution (at the same tA) this limiting value was reached at a concentration of about 2 pg ml-’ and

-loo& 0 10 20 30 40

DNA concentration (pg ml- 1)

3

Fig. 3. The dependence of the heights of AdTS ax. voltammetric

peaks 1 (a) and 3 (b) on the concentration of SC (e.0). lin (&A)

and den (m,U) plasid pBSK(-) DNA. (-,A,=), DNA was adsorbed from a 5 ~1 drop of solution without stirring;

(0,A.D). DNA was adsorbed from 1 ml of stirred solution.

Insets, plots of data obtained in the low DNA-concentration region

(0.1 to 2.0 pg ml-‘). tA=120 s, E,=-0.6 V, for other details see

Fig. 2.

peak 3 was easily detectable at a concentration as low as about 0.1 pg ml-’ . Peak 1 showed a dependence on DNA concentration (Fig. 3(a)) analogous to that of peak 3 (Fig. 3(b)). The slopes of linear portions of the dependence of peak 1 on concentrations of lin and scDNAs were less steep than that of denDNA (Fig. 3(a)). ScDNA produced no peak 3 in the whole concentration range studied in both unstirred and stirred solutions (Fig. 3(b)). However, this DNA form

6 M. Fojta, E. Palec’ek/Analytica Chimica Acta 342 (1997) I-12

can be determined by measuring peak 1 starting from about 0.5 pg ml-’ in a stirred solution at tA= 120 s. At longer accumulation times DNA can be determined at lower concentrations (e.g. 0.3 pg 1-l of scDNA or 0.1 pg ml-’ of 1inDNA at tA=5 min in the stirred solution) using either peak 1 or 3 for their determina- tion. DenDNA at a concentration of 1 pg ml-’ (i.e. 5 ng in a 5 pl drop of unstirred solution) was deter- mined with a standard deviation of 8.5% at tA= 120 s. Using 1 ml of stirred DNA solution, 1 pg of denDNA was determined with a standard deviation of 5.7% at tA= 120 s.

3.3. Interferences

It has been shown that monomeric nucleic acid components (bases, nucleosides, nucleotides) do not interfere with the nucleic acids determination when AdTS DPV or AdTS CV are used [9,11]. Similar results were obtained by AdTS a.c. voltammetry (Table 2). Other low-molecular substances (EDTA, iron EDTA complex, sodium ascorbate, hydrogen peroxide) showed practically no influence on DNA peaks.

As previously shown [9], proteins (which are, simi- larly as nucleic acids, firmly adsorbed at the electrode) may compete with nucleic acids for the electrode surface (Table 2): presence of 1 pg ml-’ lysozyme in SC (100 pg ml-‘) and ocDNA (50 pg ml-‘) sample had no measurable effect, while 10 pg ml-’ lysozyme decreased the peak 1 of SC and ocDNA by about 16 and 33%, respectively; peak 3 of ocDNA decreased by 35% under the same conditions.

3.4. Nature of peak 3 of LinDNA

Absence of peak 3 on the voltammograms of scDNA represents a qualitative difference in the beha- viour of this form of plasmid DNA as compared to its lin, oc and den forms as well as to native and denatured chromosomal DNAs [ 16191. This difference is poten- tially useful in biochemical research and can be utilized in various ways, including the detection of chemical agents inducing breakage of the DNA sugar- phosphate backbone.

Earlier it has been shown that for the peak 3 to appear easy accessibility of bases is required so that they can interact with the electrode surface [ 16,191. If

Table 2 Infhrence of various substances present in the sample of pUC19

DNA on the DNA AdTS a.c. voltammetric peak heights

Substance scDNA ocDNA Peak 1(%)

Peak 1 (%) Peak 3 (%)

None 100 100 100

Cytosine 1 mM 99 90 93

Cytidine 1 mM 103

Uridine 1 mM 99 93 92

AMP 1 mM 100 93 94

Lysozyme 1 pg ml-’ 101 96 96 10 pg ml-’ 84 67 65

[Fe(EDTA)]*- 0.1 mM 97 93 98

EDTA 1 mM 97 91 96 Ascorbate 1 mM 95

HaOa 0.03% 104

DNA was adsorbed at the electrode from 0.2 M NaCl, 10 mM Tri-

HCl pH 7.4 containing the substances listed in the table. Heights of

the respective DNA peaks measured after DNA adsorption from the

pure buffer were taken as 100%. DNA concentration: SC -

100 ug ml-‘, oc - 53 pg ml-‘; tA=60 s; for other details see text and Fig. 2.

a DME is used only denatured single-stranded (ss) DNA (in which bases can freely interact with the environment) produces peak 3 in contrast to the linear double-stranded (ds) DNA (in which bases are hidden in the interior of the DNA double helix) showing no sign of peak 3. The same is true when the HMDE is used and the potential is scanned from negative to positive values. With the same electrode, however, peak 3 appears if the potential is scanned in the usual way, i.e. from positive to negative values (e.g. Fig. 2(b)). This behaviour of dsDNA (observed at neutral and weakly alkaline pH) has been explained by a relatively slow unwinding of the DNA double helix at the electrode surface in a narrow potential range around - 1.2 V (against SCE, region U) [8,16,20]. This unwinding is manifested by both far- adaic and tensammetric signals characteristic for ssDNA [ 161 (Fig. 4). The height of peak 3 of 1inDNA increases as a result of the repeated potential scanning (at a low scan rate, Fig. 5(b)) and as a result of prolonged exposure of the DNA-modified electrode to the potentials of region U (Figs. 4 and 5); peak 1 decreases under the same conditions. Fig. 4(a) shows the increase of the peak 3 resulting from the exposure of dsDNA to the potentials of region U for the time t,=60 s obtained with a.c. voltammetry (slow scan,

M. Fojta, E. PaleEek/Analytica Chimica Acra 342 (1997) l-12 7

900

'P

600

W

300

b =t _._.- .-._.-. -.-._.-.ll 1oo

_-^. C’ , .4 L *-

‘CA

. ---------- +

_r’.

.... ...... 0.. . ..................................... ----u i

0 o’ .....................................

-1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2

EB WI

Fig. 4. Effect of the electrode potential on DNA voltammetric response. (a), AdTS a.c. voltammograms of 100 pg ml-’ SC (I) and 55 pg ml-’ lin (II) pUC19 DNA. DNA was adsorbed for r,=60 s (open current circuit, without stirring); after the transfer, potential Ea was applied prior

to the voltage scan for the time tB=60 s. I&=-O.6 V (dashed) or -1.2 V (solid), I$=-0.6 V. (c), dependences of the heights of DNA

voltammetric signals on the potential EB. Curves i and ii (right y-axis): conventional LSV peak CA (taken from [8]) of native and denatured

calf thymus DNA, respectively; ta=lOO s. AdTS a.c. voltammetric peak 1 (A,o) and 3 (A,*,+) of pUC19 DNA. (O,e), 100 pg ml-’

scDNA; (A,A), 55 pg ml -’ 1inDNA; (+), 28pgml-’ denDNA (left y-axis). U, potential region where DNA is slowly unwound at the

electrode surface. Conditions as in (a). In LSV, scan rate 0.2 V s-‘; for other details see Fig. 2.

20 mV s-l) and linear sweep (LS) voltammetric (scan rate 0.2 V s-l) peak CA (faradaic peak; data taken from [S]) on the potential EB to which DNA was exposed at the electrode for the time ta=lOO s are shown. These results show that in the a.c. voltam- metric experiment substantial part of the surface DNA unwinding occurs during the slow potential scanning, during which the DNA is exposed to the potentials of regions U for >10 s.

3.5. Supercoiled DNA is not unwound at the electrode sullface

In contrast to lin and oc forms of plasmid DNA exposure of scDNA to the potentials of region U does not result in an appearance of peak 3 suggesting a significant unwinding of DNA (Figs. 4 and 5), which is in a good agreement with the results of CV studies [ 111. A very small peak 3 produced by scDNA as a result of prolonged exposure to the potentials of the region U (Fig. 4) or resulting from the repeated

potential scanning (Fig. 4) might be due to traces (<5%) of oc and/or 1inDNA in the scDNA sample hardly detectable on the agarose gel (Fig. l(b) and (c)). [Addition of 5% of 1inDNA to the scDNA resulted in a peak 3 already on the first scan; after the second scan this peak was substantially higher than that of scDNA alone (Fig. 5(b))]. On the other hand it cannot be excluded that the prolonged exposure of scDNA to the potentials of region U might result in a very limited local unwinding of some small parts of the molecule of scDNA.

3.6. DNA nicking by y-irradiation in solution

DNA is nicked due to ionizing irradiation [21,22]. Supercoiled pUC19 DNA at a concentration of 120 pg ml-’ (in 0.2 M NaCl, 10 mM Tri-HCl pH 7.4) was irradiated with a 6oCo y-source. DNA samples were exposed to irradiation by doses ranging from 0 to 150 Gy. As a result of the irradiation (dose 16 Gy), a band of ocDNA appeared on agarose gel

M. Fojta, E. PaleEek/Analytica Chimica Acta 342 (1997) 1-12

9001

‘P .

6co-

-1.5 E(V) -0X

Fig. 5. (a) The dependences of the heights of AdTS a.c. voltammetric peak 3 of pUCl9 DNA on the time ta, for which the immobilized DNA

was exposed to the potential Ea. (O,+), 100 pg ml-’ scDNA; (0.x). 55 pg ml-’ 1inDNA; (A), a mixture of 100 pg ml-’ SC and 15 pg ml-’

1inDNA (B), 28 pg ml-’ denDNA. (+,x), Es=-0.6 V; (0,&n,=), Ea=-1.2 V. (b) The effect of repeat voltage scan on the voltammetric

response of pUC19 DNA-modified HMDE. AdTS a.c. voltammograms of (I), scDNA (100 pg ml-‘); (II), 1inDNA (55 pg ml-‘); (III), mixture of SC and 1inDNA (100 pg ml-’ and 15 pg ml-‘, respectively). Dashed, first scan (from -0.6 to -1.5 V); solid, second scan of the same DNA- modified electrode. For details see Figs. 2 and 3.

(Fig. l(c)). The dose of 150 Gy almost completely changed scDNA into the oc form (Fig. l(c), track 4). AdTS a.c. voltammetry of the respective samples showed significant increase of peak 3 with the dose (i.e. with the increasing amount of nicked DNA in the sample); the peak lc increased only slightly (Fig. 6(b)).

Compared to gel electrophoresis the determination of y-radiation-induced DNA nicking by AdTSV is faster and the quantitative evaluation of the voltam- metric signals is easier than that of ethidium bromide- stained gel bands. The sensitivity of the electroche- mical determination can be greatly increased by brief heating of the irradiated DNA at 96°C which turns every nicked (but not SC) DNA molecule into a dena- tured form [3] (Fig. l(a)) producing a high peak 3 (Fig. 6(a)). [On the agarose gel, the ocDNA band disappeared as a result of its denaturation (Fig. l(c)).] In this way less than 2% of nicked molecules i.e, less than one scission per >2.5 x lo5 phosphodiesteric bonds can be determined. [A small amount (~2%) of nicked DNA in the control sample was detected after heating (Fig. 6(a)); band of this DNA appeared

on the 1 O-fold overloaded agarose gel (Fig. l(c), track 5)]. The sensitivity of this determination is very much dependent on the purity of the scDNA sample. Higher content of oc or 1inDNA in the sample would result in a higher background and decrease in the sensitivity of the method.

3.7. Cleavage of DNA by hydroxyl radicals in

solution and at the electrode surface

A number of natural and synthetic reagents have an ability to bring about the DNA strand cleavage [23]. Among them the hydroxyl radical has several features which distinguish it from other DNA cleavage agents [15]. This radical is a small entity compared to the protein molecules of deoxyribonucleases and even the chemical nucleases methidiumpropyl/EDTA. Fe(I1) and bis- l,lO-phenanthroline copper. The hydroxyl radical is highly reactive showing no preference for cleavage of a particular base. It has been used mainly for high resolution footprinting of DNA protein com- plexes [24]. We generated hydroxyl radicals by the Fenton reaction of hydrogen peroxide with the

M. Fojta, E. Pale~ek/Analytica Chimica Acta 342 (1997) l-12 9

IP

(n7

100

E

b 0

0 100 200

dose (Cy)

Fig. 6. Nicking of SC pUC19 DNA by y-irradiation. (a,b), the dependence of heights of the AdTSV peak 3 (A& and peak 1 (0) on the

radiation dose: (a), at lower and (b), at higher doses. (A.0). unheated samples; (A), samples heated after irradiation to 96°C for 5 min and

quickly cooled. For calibration, known amounts of denatured ocDNA were added to the unheated scDNA samples (horizontal dotted lines: heights of peak 3 obtained as a result of the presence of(i) 1.5%, (ii) 5% and (iii) 10% of denDNA in scDNA). (c) AdTS a.c. voltammograms

of control scDNA sample (I) and DNA irradiated with a dose of 8 Gy (II); solid, heated samples; dashed, unheated. For other details see Fig. 2.

complex of Fe(I1) and EDTA:

[Fe(EDTA)]‘- + Hz02 + [Fe(EDTA)]-

+ OH- +’ OH.

An electron from the Fe(I1) split hydrogen peroxide into a hydroxyl radical and the hydroxide ion. Ascor- bate is added to reduce iron(II1) to iron( which then reacts with more hydrogen peroxide. The hydroxyl radical cleaves DNA by abstracting a hydrogen atom from a sugar residue in the DNA backbone. The deoxyribose residue breaks down, leaving a gap with predominantly 3’- and 5’-phosphate ends; a small amount of phosphoglycolate is also formed.

3.7.1. Cleavage of DNA in solution Samples of pUC19 DNA were treated with the

reaction mixture (see Section 2) for 1 min. Then the HMDE was immersed into a drop of the sample followed by the usual AdTSV procedure. As a result of nicking and further degradation of the plasmid

DNA (observed by agarose gel electrophoresis, Fig. 7(b)), the peak 3 appeared (Fig. 7(a)) and increased with increasing concentration of the iron complex (the dependence was approximately linear from 1 to 50 PM, Fig. 7(c)). Similarly to the y-irra- diation, only small changes of the height of peak 1 were observed under these mild conditions (up to 5 PM [Fe(EDTA)12-). At higher concentrations of [Fe(EDTA)12- (lo-25 PM) when larger number of strand breaks accumulated per DNA molecule, the latter peak increased by about 65%, reaching the height observed with 1inDNA (this is in agreement with the gel electrophoresis, which showed a band of linDNA, Fig. 7(b), track 4). Up to 100 pM [Fe(EDTA)12-, further slight increase of the peak 1 was observed. Under strong reaction conditions, both peaks (1 and 3) were higher than those of thermally denatured plasmid DNA suggesting that the DNA cleavage resulted in production of short DNA frag- ments (showing a smear on the gel electrophoresis, Fig. 7(b)).

10 M. Fojta, E. PaleEek/Analytica Chimica Acta 342 (1997) 1-12

1000

I3

500

b9

I I -1.5 E(V) -0.6

b 1234567

0 20 40 60 80 100 120

concentration of [Fe(EDTA)]z-(PM)

Fig. 7. (a) (I), AdTS ax. voltammograms of SC pUC19 DNA; (II), curve of the same DNA treated with 10 pM [Fe(EDTA)]‘- reagent for

1 min in solution (see Section 2) prior to adsorption at the HMDE; (III), curves of the same DNA treated with the above reagent for 1 min at

the electrode surface (after the DNA adsorption from solution not containing ihe reagent). Solid curves, fast scan; dashed, second scan (curve

II, first scan only). (b), 1.2% agarose gel electrophoresis of pUC19 DNA (450 ng per track) treated with the [Fe(EDTA)]‘- at concentration of

0 (track l), 1 PM (track 2). 5 pM (track 4), 25 pM (track 5), 50 pM (track 6) and 100 PM (track 7) for 1 min. (c), the dependences of the

height of peak 3 of the DNA on the [Fe(EDTA)]*- concentration. (O), cleavage in solution; (&A), cleavage at the electrode; (A,o), first scan; (A), second scan. Other details as in Fig. 2.

3.7.2. Cleavage of DNA at the electrode sutiace followed by washing and AdTSV measurement in Intact SC pUC19 DNA was immobilized at the the blank electrolyte. Peak 1 and peak 3 increased

HMDE in the usual way, under conditions securing with increasing concentration of the reaction mixture full coverage of the electrode. The DNA-modified (Fig. 7(c)). Compared to DNA cleavage in solution electrode was then washed and immersed in the (Fig. 7(c)) the increase of peak 3 was steeper up to reaction mixture (not containing DNA) for 1 min, 5 pM [Fe(EDTA12- but at higher [Fe(EDTA)12- con-

M. Fojta, E. PaleEek/Analytica Chimica Acta 342 (1997) 1-12 11

centrations a decline from linearity was observed. At 50 uM [Fe(EDTA)12- peak 3 and peak 1 reached their limiting values, in contrast to the DNA cleavage in solution where both peaks increased with increasing [Fe(EDTA)12- concentration up to the highest (100 PM) concentration of [Fe(EDTA)12- used. These results show that in the initial stages of the reaction (i.e. under conditions when the number of the strand breaks is relatively low producing oc and 1inDNA molecules as the main products) the rate of cleavage of DNA immobilized at the electrode does not substan- tially differ from that observed in solution. In further stages the cleavage of DNA at the electrode is almost stopped, while the rate of cleavage of DNA in solution is only slightly decreased (under the same bulk con- centration of the reaction mixture). Earlier we have observed [25] a similar inhibition of enzymatic (ribo- nuclease) cleavage of RNA immobilized at the elec- trode. We tentatively suggest that the inhibition of cleavage of DNA at the electrode at higher [Fe(EDTA)12- bulk concentration (Fig. 7(c)) or longer cleavage times might be at least partly due to a decreased accessibility of the sugar phosphate backbone anchored at the surface to the ‘OH radicals. It has been shown [26] that DNA adsorbed at the flat calcium phosphate surface is cleaved by deoxyribo- nuclease I less effectively at the phosphodiesteric bonds located close to the surface. This principle has been utilized as a general method of DNA foot- printing in which binding of DNA to some material (e.g. protein) protects the DNA molecule from clea- vage by an enzymic or chemical agent at particular sites [24].

4. Conclusions

Electroactivity of nucleic acids was discovered more than 3.5 years ago [27,28]. Electrochemical methods provided an early evidence of the DNA premelting and polymorphy of the DNA double helix (221. Now it becomes increasingly clear that the mercury and carbon electrodes in combination with modem voltammetric and potentiometric methods represent versatile and highly sensitive tools in nucleic acid and molecular biological research [ 121. Sensitive determination of RNA in DNA samples [9,29], studies of helix-coil transitions in different forms of plasmid

DNA [l 11, interaction of DNA with antineoplastic drugs in solution and at the electrode ([30], E. PaleEek and P. Perez, unpublished), determination of nucleic acid components at nanomolar concentrations by CSV [31], chronopotentiometric analysis of RNA and plas- mid DNA with carbon electrodes [32,33] (potentially useful in DNA hybridization analysis) can be men- tioned as examples of the recent studies based on the measurements of the intrinsic electrochemical signals of the nucleic acids.

In this paper we show that different forms of plasmid DNA are producing ac. voltammetric peaks of different intensity and at different potentials. Absence of peak 3 in covalently closed circular scDNA and the appearance of this peak as a result of a single interruption of the sugar-phosphate backbone in this DNA (Figs. 2, 6, 7) has been utilized for the determination of the DNA damage. If scDNA is immobilized at the HMDE a new versatile biosensor is created which can be applied for numer- ous purposes in biochemical and environmental research. Advantages of this sensor include fast responses, high sensitivity, relatively low cost, etc. In difference to the methods detecting presence of known mutagenic and other agents damaging DNA, this sensor is capable to detect any agent interrupting the DNA strands. Some agents are known to induce DNA damage (e.g. chemical modification of bases) without introducing DNA strand breaks. In many cases such a damage results in labilization of the sugar-phosphate backbone which can be trans- formed into strand breaks by some treatment such as heating at alkaline pH. More details will be published elsewhere.

It has been shown that carbon electrodes in combi- nation with constant current chronopotentiometric stripping analysis (PSA) can produce well developed DNA and RNA oxidation signals [32-341 at nucleic acid concentration bellow 1 ug ml-‘. Application of PSA in combination with solid electrodes to nucleic acids represents a new and important development in their analysis. PSA, however, is much less sensitive to differences in the forms of plasmid DNAs [33] and can be therefore hardly applied for a sensitive detection of interruptions of the DNA sugar-phosphate backbone. Nevertheless, both carbon and mercury electrodes can be applied for the studies of other types of FNA damage, including the damage to DNA bases and

12 h4. Fojta, E. Palec’ek/Analytica Chimica Acta 342 (1997) 1-12

covalent binding of mutagenic and carcinogenic sub- stances to DNA.

Acknowledgements

The work was supported by grants from the US- Czechoslovak Science and Technology Program Reg. No. 930 11, and the Grant Agency of Academy of Sciences of the Czech Republic Reg. No. 204/97/ K084. The authors thank Dr. Aiena Kuderovi and Dr. Frantigek Jelen for critical reading of the manu- script.

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