Surface enhanced raman scattering spectroscopy of methylated guanine and dna

Surface Enhanced Raman Scattering Spectroscopy of Methylated Guanine and DNA* J E A N - M A R I E St~QUARIS, J O A N N E FRITZ, H E L M U T LEWINSKY, AND E C K H A R D K O G L I N Institute of Applied Physical Chemistry, Nuclear Research Center (KFA), D-5170 Juelich, Federal Republic of Germany

Received October 10, 1984; accepted November 19, 1984 Surface enhanced Raman scattering (SERS) spectroscopy provides a new method of identifying nucleic bases in low concentrated solutions after adsorption on silver colloids. In particular, highresolution SERS spectra of mutagenic effectscausing methylatedderivativesof guanine at concentrations as low as 10-4 M can be obtained. The absence of a carbonyl Raman band in the SERS spectra of 3methylguanine and 9-methylguanine suggests specific chemical species or orientations at charged interfaces. Observing SERS spectra of methylated DNA at a silver electrode, the major product of alkylation, 7-methylguanine, can be detected. In addition conformational changes of methylated DNA according to the elucidated sequence of induced short-term and long-term damages can be probed. © 1985 Academic Press, Inc.

INTRODUCTION Modified nucleic bases can occur naturally in various nucleic acids (1). However, exogeneous alkylation of cellular nucleic acids by environmental chemicals appears to be a prerequisite for the action of certain chemical carcinogens (2). Detailed analyses of the nucleic bases of D N A and RNA show that the major product of the reaction is 7-alkylguanine (2, 3). However, different reports (4, 5) have established that m i n o r products of the alkylation also have relevance to mutagenesis or carcinogenesis. Highly sensitive methods are thus required for the detection of alkylated products in the nucleic acids. In previous work (6, 7), preliminary results obtained by surface enhanced raman scattering (SERS) spectroscopy have shown that nucleic bases adsorbed at silver colloid surfaces give rise to intense R a m a n signals. To test the sensitivity and selectivity of the SERS detection, applications of this new method have been

* This paper is dedicated to Dr. Milton Kerker on the occasion of his 65th birthday.

carried out with methylated derivatives of guanine (see Fig. 1). It is shown that wellresolved vibrational spectra of guanine derivates at concentrations as low as 10 .6 M can be obtained. The R a m a n spectrum of 0 6methylguanine, a direct marker of carcinogenic chemicals (4, 5) is thus reported for the first time. Furthermore, the variations in structure of methylated D N A adsorbed at a silver electrode have been followed by a spectroelectrochemical method. The development of this method, suitable under physiological salt conditions, has brought much insight into the effects of 3,-irradiation (8) and antitumoral N-coordination c o m p o u n d complexation (9) with DNA. Also, short- and long-term damaging effects of the D N A structure by methylation have been probed. The results obtained with SERS on the behavior of native, methylated, and irradiated D N A adsorbed at charged interfaces agree with previous studies in voltammetry (12, 33, 37). Both methodologically independent approaches support each other. Potentialities and essential findings obtained by voltammetry on the interfacial behavior and decon-

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418

SI~QUARISET AL. 0 II

R 2

0

II 8 R2

I : R1 =H, R2=H I1: Rl =CH3, R2=N II1: F~ =H, R2=CH2

O/

IV:

71qeGua

CH3 Gua 1MeGua 9f4e Gue

ICH3

v: 3Ne Guct

I~3

Vl : R3= OCH3 VII :R3= 01

06 NeGua 6 CI Gua

FIG. 1. Derivates of guanine. (I) guanine (Gua); (II) 1-methylguanine, 1-MeGua; (III) 9-methylguanine,9MeGua; (IV) 7-methylguanine,7-MeGua; (V) 3-methylguanine,3-MeGua;(VI) Or-methylguanine,Or-MeGUa~ (VII) 6-chloroguanine,6-ClGua. formation have been summarized in a recent review (40). MATERIAL AND METHODS

Chemicals. Guanine (Gua), 1-methylguanine (1-MeGua), 3-methylguanine (3MeGua), 7-methylguanine (7-MeGua), and 9-methylguanine (9-MeGua) were obtained from Fluka A.G. and 2-amino-6-chloropurine or 6-chloroguanine (6-C1Gua) from Sigma Chemical Company. Calf thymus (CT) DNA (MW = 2 × 106) was purchased from P L Biochemicals. Dimethylsulfate was obtained from Fluka A.G. O6-Methylguanine (0 6MeGua) was prepared from 6-chloroguanine according to Balsinger and Montgomery (10). All other chemical reagents were of analytical quality and were purchased from E. Merck. Methylation ofDNA. A solution of 1 mg/ ml of CT DNA in 0.5 M Tris-HCl buffer (pH 8) was methylated after incubation for 1 h at 37°C in the presence of 3% dimethylsulfate. Subsequently the solution was dialyzed against 0.1 M KC1, 2 X 10-3 M TrisHC1 buffer (pH 8) at 5°C. The determination of the percentage of 7-MeGua has been determined electrochemically as described elsewhere (11). The depurination of methylated Journal of Colloid and Interface Science, Vol. 105, No. 2, June 1985

DNA was obtained by incubation of the final solution of methylated DNA at 70°C during 3 h (12). Silver colloid preparation. Silver colloids were prepared by reduction of AgNO3 with NaBH4 (13). In a typical experiment, three volume parts of 2 x 10-3 M NaBH4, cooled in an ice bath, are added dropwise to one volume part of 10-3 M AgNO3 and mixed vigorously. The obtained yellow solution has an absorption maximum at 380 nm which corresponds to an average particle diameter o f 42 nm according to Kerker et al. (14, 15). The silver colloid solution can be stored at 5°C in absence of salts for weeks without any changein color. The adsorption of guanine and its derivates is performed by addition of aliquots containing a solution of 5 mg/liter guanine in 5 X 10-3 M HC1 to the silver colloid until a change in the original yellow color is observed. Indeed the shift of the absorption maximum of the silver colloid solution to longer wavelengths indicates increasing size or deformation of the original spherical silver particles (13, 14, 15) induced by the adsorption of nucleic bases. The final concentration of guanine and its derivates in silver colloid solution ranges from 10 -6 tO 10-5 M at a pH of 4.5. Raman spectroscopy. Surface Raman spectra were measured using a computer-controlled double monochromator (Spex, Model 14018) with a cold photomultiplier (RCA 31034 A), operated in thephoton-counting mode. Monochromator slits were selected to provide a better than 8-cm-1 bandpass. The excitation wavelength was the 514.5nm line of an argon ion laser (Spectra Physics, Model 164.06). Laser powers at the sample were respectively 5 and 100 mW for the silver electrode and the colloidal Ag particles. The spectra were recorded with the Spex 1459 UVISIR illuminator in a 1-ml liquid cell (colloid-SERS spectroscopy) or in a spectroelectrochemical cell (electrode-SERS spectroscopy). Further details of Raman instrumentation are given elsewhere (16).

SERS OF METHYLATED DNA

419

1-MetlwIgJaninelag Eoltoid

3 q"lethytguanlne/ Ag Cottoid -i

oo

~5oo '

Raman shill ~ (cm-1)

12'oo

9bo

6bo

~o

Raman shg:L? (/~I)

FIG. 2. SERS spectrum of 1-methylguanine (II) adsorbed on Ag colloids. C-3.3 X 10-6 M, pH 4.5.

FIG. 4. SERS spectrum of 3-methylguanine (V) adsorbed on Ag colloids. C-8 X 10 ~6 M, pH 4.5.

Spectroelectrochemical method. The spectroelectrochemical apparatus and procedure have been previously described (16). However, the prerequisite etching procedure of the silver surface (17), analogous in principle to the above-described silver colloid microstructure production, has been modified. The electrolysis current (1 mA) during the oxidation and the reverse reduction of the silver electrode is now imposed during a cycling time of 10 s. The procedure was repeated three times for the D N A solution from the adsorption potential Es - 0.2 V vs Ag/AgC1 (reference electrode). This new procedure improves the standardization of the method.

RESULTS AND DISCUSSION

[] ~

L S E R S Spectroscopy of Guanine and Its Derivates at Silver Colloids Figures 2, 3, 4, and 5 show typical SERS R a m a n spectra of methylated guanine derivates (II, III, V, VI) adsorbed at silver colloids. The substantially higher sensitivity of this method by a factor 105-106 in comparison with the normal R a m a n scattering (NRS) spectroscopy (13, 14, 15) permits rapid spectra recordings with a conventional R a m a n spectrometer of biomolecules at low bulk concentration down to 10 -6 M. The observed frequencies and relative intensities of the SERS

9-MefhyLguanine/Ag[oltoid 05q'lefhytguanine/~ Coltoid E

15()0

12~) c~O Roman shift ~ (crn~1)

660

300

FIG. 3. SERS spectrum of 9-methylguanine (III) adsorbed on Ag colloids. C-1.4 X 10-6 M, pH 4.5.

15'00

I~0 900 Raman shift~ (cm-1)

600

300

FIG. 5. SERS spectrum of Or-methylguanine (VI) adsorbed on Ag colloids. C-4 X 10 -6 M , pH 4.5. Journal of Colloid and Interface Science, Vol. 105, No. 2, June 1985

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SI~QUARIS ET AL.

b a n d s o f g u a n i n e a n d its d e r i v a t e s a r e g i v e n i n T a b l e I. M o s t o f t h e b a n d s i n S E R S spectra are very similar to their known NRS c o u n t e r p a r t s (18, 19). T h e l a r g e s t f r e q u e n c y s h i f t is a b o u t 3 0 c m - I . H o w e v e r it m u s t b e remarked that SERS spectra of nucleic bases are obtained in their neutral forms while the already published frequencies by NRS spect r o s c o p y (18, 19) o n l y c o n c e r n t h e i o n i z e d forms in solution. Indeed, in the case of NRS spectroscopy, the low solubility of guanine

a n d its d e r i v a t e s r e q u i r e s e x t r e m e p H v a l u e s to establish the then necessary bulk concent r a t i o n r a n g e f r o m 10 -2 t o 10 -~ M . A c o m m o n f e a t u r e o f t h e S E R S s p e c t r a is the Raman band in the low-frequency region representing specific interaction between adsorbed molecules and the silver metal. Although the highly positively charged surface o f s i l v e r c o l l o i d s (20, 2 1 ) f a v o r s a s p e c i f i c adsorption of chloride ions, the rather weak R a m a n signal o f t h e a d s o r b e d C 1 - / s i l v e r m e t a l

TABLEI SERS Fr~uencies(cm-l) ofGuanine DefivatesAdsor~datSilverColloids G u a (I) C = 3.6 X 1 0 ~ M

234 336 380 455 506 552

v w w w w w

l-MeGua (I!) C = 3.3 X 1 0 ~ M

232 326 366 476 526

v w w w w

9-~'eGua (I11) C = 1.4 × 10-6M

7-MeGua ( 1 ~ C = 4.2 × 1 0 ~ M

3-MeGua (V) C = 8.4 × 10-rM

223 v

232 v

222 v

361 w 495 w 522 w

396 w 455 w 505m 559 w

368m 462m 501 w 559m

651 v 703 v

638 v 735 m 765m

Or-MeGua (VI) C = 4,5 × 10-rM

224 339 395 429

v w w w

570 w

607m

6 ~ t G u a (VII) C = 5 × 10-rM

236 v 368 w 452 w 536 w 582 s

620 v 653 v

1054 w

655 714 762 814 938 978 1028

1145s

1147m

745 w 856 w 964 m

v w w w m w w

1224m 1260 w 1317 w

1231 s 1291 m

1356 s 1386v

1356 s 1375m 1405m 1460m

656 s 730 w

752 w

871 w 911 m 1047m 1092 w 1200 w 1235 w 1285m

1139s 1183 s 1218m 1272 s

921 w 995 w 1055m

946 w

1154m

1134w

1221 s 1243 v 1285 s

1240 w

752 844 938 963

w w w w

1300 s

1467 v 1512m 1538 s 1574m 1594m 1655m 1708 s

1335 v 1396m

1354 v

1446m 1498m

1421 s 1462m 1495 s

1580 s

1565 s

1349 s 1387 v 1421 v

1334 v 1386m

1384m

1510m

1506 w

1496 w

1549m 1589 w

1592 s

1608 w 1628 w

1700 s

1703 v

Note. w, m; s, v indicate weak, medium, strong, and very strong intensities, respectively. All reported Raman frequencies in cm -1 have a precision within +4 cm -~.

Journal of Colloid and Interface Science, Vol. 105, No. 2, June 1985

SERS OF METHYLATED DNA

421

at 245 cm -~ in the absence of guanine suggests this relatively low-frequency band to N-CH3 other silver surface interactions to explain deformation modes is not reasonable (23). the high intensity and broadness of the Ra- Only a coupling of ring vibrations with N7man bands in the 200-cm -~ region. As re- CH 3 could explain this Raman band. Another striking feature of the SERS specported by several authors, direct interaction of the metallic silver with atoms in covalent tra of N-methylated guanines is the depenbonds also gives rise to specific vibration dence of the carbonyl stretching vibration on modes (22, 23). A possible overlapping of the site of methylation. In Table I, it is the SERS bands thus diminishes the resolu- observed that SERS spectra of 9-MeGua (II) tion of the spectrum by an enlargement of and 3-MeGua (V) depart substantially from the SERS signal. However, in the case of the spectra of l-MeGua (II) and 7-MeGua guanine and its methylated derivates, it is (IV) in the 1700 cm -1 region. Failure to proposed in reference to pyridine and 2,6- observe the C6=O stretching vibration frelutidine that the low spectral band at 230 quency at pH 5 for both neutral forms of cm-I can be predominantly attributed to Ag- methylated derivates of guanine (III and V) N stretching vibrations (23). The other more can be interpreted differently. A first suggesintense bands in the SERS spectra of the tion would assume a tautomeric form of guanine are due to breathing modes in the guanine at neutral pH. Indeed the SERS 600-cm -~ region and to coupled ring and spectra of III and V (Figs. 3 and 4) may be double bond stretching vibrations in the re- interpreted by a displacement of the tautogion 1500 to 1700 cm -~. Some ring vibrations meric equilibrium from the predominant keto are also very pronounced in the interval 1200 form in solution --N1H--C6=O to the enol to 1500 cm -~. These Raman bands, boldface I in Table I, are thus of interest in detecting form --NI=C6--OH in the adsorbed state. modified nucleic acid bases by the SERS I method. For example, the ring breathing This hypothesis is supported by the comparvibration of guanine (I) at 653 cm -~ is shifted ison of the SERS spectra of 1-MeGua (II) to shorter frequencies by substitution of the and Or-MeGua (VI) in the carbonyl stretchoriginal carbonyl group C6=O with ing vibration region (Figs. 2 and 5). The law of valence requires in fact that methylation C 6 - - O C H 3 ( V I ) t o 620 cm -~ and with C6C1 at the 0 6 (VI) and N~ (II) locks respectively (VII) to 582 cm -~. Such Raman shifts obviously agree with a mass effect but they also the enol and keto forms of the guanine ring. confirm the strong coupling effect of the The absence of the C6=O band in SERS carbonyl vibrations to the ring stretching spectra of 9-MeGua (III) and 3-MeGua (V) vibration of the purine cycle (24). Further- (Figs. 3 and 4), and as expected in 0 6more, supporting evidence for the assignment MeGua (Fig. 5), would thus suggest their of the carbonyl stretching vibration to the enolic forms. On the other hand, Gua (I) intense Raman band near 1700 cm -1 (18, and 7-MeGua (IV) preserve their keto form 19, 24) in the SERS spectrum of guanine is in the adsorbed state just as 1-MeGua (II) provided by its absence from the spectra of (Fig. 2). An alternative structure to this rare O6-MeGua (VI) and 6-C1Gua (VII) (Table I tautomeric form at neutral pH would involve and Fig. 5). The fixation of the methyl group a deprotonation of the proton in position NI at N positions of the purine cycle also con- for the adsorbed methylated derivates (III siderably modifies the SERS spectrum of and V). Indeed the monoanionic form of 9guanine. For example, an additional very MeGua (III), --N~ =C 6-(5 in basic solution, strong Raman band is seen at 703 cm -1 of I 7-MeGua (IV). However, the assignment of does not show any Raman band above 1570 Journal of Colloid and Interface Science, Vol. 105, No. 2, June 1985

422

SI~QUARIS ET AL.

cm -1 (19). When the hydrophobic (26) and electrical properties do not favor enolization and ionization of C6=O then an orientation effect should be considered. Indeed, another possible interpretation is based on the shortrange effects (27, 28) of the SERS enhancement factors and on specific orientations of the guanine derivates at the silver surface. As the electromagnetic model predicts (29) a very rapid decrease of SERS effects with distance on the A scale would limit the enhancement factors to bond vibrations in the immediate vicinity of the silver surface. The disparition of the carbonyl stretching vibrations in SERS spectra of 3-MeGua (V) and 9-MeGua (III) would thus suggest that the C6----O bond lies far away from the surface. The hypothesis of an "inactive" SERS carbonyl vibration would consider orientations for both methylated guanines (III and V) in the adsorbed state different from the other derivates. However, results of a normal coordination calculation of the guanine cycle (24) show that the 1700-cm -1 Raman band involves coupling ring and carbonyl vibrations. The rather complex character of this band thus moderates the exceedingly high specificity of the particular adsorption behavior for III and V. From the preceding interpretations it follows that SERS spectroscopy can give insight on the interfacial behavior of molecules. Further progress in the theoretical treatment of the SERS phenomenon woul~l help to clarify further the assignments of characteristic bonds, Otherwise, similar SERS characteristics observed in the nucleotidic derivates of guanine as in 9-MeGua will necessitate further investigations to establish definitively the Carbonyl structure of the C6z O bond for nucleic acids in comparable interfacial conditions~ However, specific Raman bands for the guanine ring vibrations, less sensitive to interfacial events, can be used for characterization purposes as the detection of the sites of alkylation in DNA. It can also be added that after an application of the silver colloid solution of methylated guanines on silica gel Journal of Colloid and Interface Science, Vol. 105,N,6. 2, June 1985

plates, used for thin layer chromatography, SERS spectra at the nanogram and subnanogram level are recorded (42).

II. SERS Spectroscopy of Methylated DNA at the Silver Electrode The reaction of DNA with alkylating agents results in the formation of alkylated purine bases within the DNA macromolecule (2-5). The conversion of Gua (I) into 7-MeGua (IV) is the major effect of monofunctional methylating agents such as dimethylsulfate (3, 12). These alkylated sites in DNA are very unstable and leave apurinic sites which are, in turn, hydrolyzed to give as a final product a DNA molecule with strand breaks (12, 30). The successive events, generally considered to be important in the mutagenic and carcinogenic activities of alkylating agents (2) have been established and confirmed by voltammetric studies at the mercury electrode (12). In a similar way, SERS spectroscopy has been employed at a silver electrode to probe the effects of the methylation on the DNA structure. Figure 6 shows the SERS spectra of native and methylated DNA adsorbed at a silver electrode. Before discussion of the specific changes in SERS spectra due to the methylation, it must be remembered that the Raman band assignment only involves the adsorbed parts of DNA at the electrode surface (8, 31). At an adsorption potential o f - 0 . 2 V which corresponds to a high positively charged surface (32), the SERS spectra of adsorbed nucleic acids are characterized by a strong Raman band at 236 cm -l (Fig. 6A). This SERS specific low-frequency vibration, already mentioned in the case of silver colloids, has been attributed here to an electrostatic interaction of negatively charged phosphate groups with the silver surface. It is seen that after reaction of the alkylating agent with DNA the 236-cm -l band decreases (Fig. 6B). As is seen in Fig. 6C the band at 236 cm -1 corresponding to the interaction of the phosphate with the silver surface is substantially decreased for methylated DNA in the stage

SERS OF METHYLATED DNA

o~ o ~o-*

~.o!

~o

~

t--.

~

o

1go

os o

sgo

o

J1 ,

T

/ ~

w

9go

13'5o (cm-l')

FIG. 6. SERS spectra of native CT DNA (A) and of methylated CT DNA (8% 7-MeGua)before(B) and after (C) heating of 70°C adsorbed on Ag electrode. Concentration of DNA 200 ~g ml-~, 0.15 M NaC1, 2 × 10-3 M Tris, pH 8, adsorption potential E~ - 0.2 V vs Ag/AgCI (referenceelectrode). of hydrolytic release of 7-MeGua from the strand. This intensity change can be interpreted as a decrease of the adsorption of modified DNA through the nucleic phosphate groups. As we have shown previously by studies with voltammetry and SERS (8, 12, 33), the destabilization of the double-stranded structure of DNA by damages induced by ionizing radiation or mutagenic chemicals

423

brings the inner-lying nucleic bases in contact with the surface enabling direct measurements by SERS and voltammetric methods, of responses due to the interaction of the bases with the charged interface (8, 12, 33, 37, 40). The results presented in this paper give added information on the variations of adsorption sites from DNA after chemical modification. Additional support for this fact is given by the examination of the other SERS bands of native and methylated DNA (Figs. 6A and B). In the SERS spectrum of native DNA, the Raman bands at 736 and 1332 cm -1 are assigned to adenine residues from adsorbed regions of DNA (31). It has to be remarked that, depending on the electrochemical pretreatment of the silver electrode and the available quality of the DNA samples, the Raman intensity of the adenine vibration can vary somewhat. Another Raman band sensitive to the sugar-phosphate backbone conformation of DNA is localized in the 800-cm -1 spectral region (31, 34, 35, 36). However, the assignment of the SERS band at 794 cm -1 to a simple vibration is not evident. It has been established that, due to the adsorption forces and according to the interfacial electric field, unmodified native DNA is also destabilized in its helical structure (37). After comparison of SERS spectra of native DNA with various nucleic base compositions, it appears that a possible overlapping of cytosine or thymine ring modes and phosphate diester stretching vibrations obscure the spectral resolution in the region 760-830 cm-L This notwithstanding, the effects of chemical methylation lead to specific variations in the SERS spectrum (Fig. 6B). The SERS spectrum of methylated DNA shows new Raman bands at 656, 700, and 1360 cm -~ which correspond to characteristic vibrations of the 7-MeGua (IV) residues in adsorbed methylated DNA (viz., Table I). Furthermore, the decrease upon methylation (Fig. 6B) of the band at 1200 cm -1 and the 1300-cm -~ shoulder of the band centered at 1332 cm -~ in the SERS spectrum of native Journal of Colloid and Interface Science,

V o l . 105, N o . 2, J u n e 1985

424

SI~QUARIS ET AL.

DNA (Fig. 6A) can be related to a conformational change of modified nucleic base pairs 7-MeGua-cytosine. Direct evidence for the assignment of both bands to adsorbed cytosine residues has been obtained from SERS results with polynucleotides containing cytosine residues (41). A relative increase of the adenine residue adsorption by sharper Raman bands at 736 and 1330 cm -1 must also be noted. Thus, at a rather positively charged surface, the SERS spectra reveal substantial changes in the adsorption behavior o f methylated DNA. In summary, the effects of the chemical methylation on DNA are manifested in SERS spectra (1) by a lower accessibility of cytosine residues counterbalanced by the adsorption of the paired 7-MeGua residues, (2) by an additional accessibility of adenine residues from chemically induced destabilization of DNA regions (12), and (3) by a concomitant decrease of the adsorbed nucleic phosphate groups. This short-time effect of the methylation is followed after longer time spans by a hydrolytic degradation of the methylated DNA. The cleavage of 7-MeGua, accompanied by strand breaks, can be rapidly obtained by heating the DNA solution at 70°C over 3 h (12). Before discussing the assignment of the Raman bands in the SERS spectrum (Fig. 6C) it must b e clearly noted that a similar heating treatment of unmethylated native DNA does not modify its SERS spectrum (Fig. 6A). By comparison the SERS spectrum of degraded methylated DNA exhibits large variations. In the studied positive adsorption potential range, it appears that most Raman bands can be assigned to vibrations of the released 7-MeGua (IV) (Table I). This rather surprising result points out the importance of the different adsorption behavior of the nucleic acid components in a solution of partially hydrolyzed DNA. Considering a competitive adsorption at the silver metallic surface, it must be assumed that the strong hydrophobic character of the 7-MeGua prevails over the more hydrophilic Journal of Colloid and Interface Science, Vol. 105, No. 2, June 1985

properties of the oligonucleotide units from the degraded DNA. The preferential adsorption of 7-MeGua permits (Figs. 6B, C) a rapid identification of the major chemical modification of DNA. It must also be noted that the sensitivity of the SERS method in detecting conformational changes in the different steps of the degradation of methylated DNA is much higher than that of NRS spectroscopy in solution (38). Although the adsorption at a metallic surface is a prerequisite for SERS, it must be recalled that interfacial conditions such as hydrophobicity or the presence of electrical charges are also encountered by DNA in much more complex biological structures in the living cell (39). ACKNOWLEDGMENTS The authors thank Dr. P. Valenta for interestingand helpful discussion, Dipl. Ing. D. Unterlugauer for the preparation of Or-methylguanine, and Prof. Dr. H. W. Nfirnberg for his continuous encouragementand critical reading of the manuscript. REFERENCES 1. Borek,E., and Srinivasan,P. R., Annu. Rev. Biochem. 35, 275 (1966). 2. Lawley, P. D., in "Chemical Carcinogens" (C. E. Searle, Ed.), ACS Monograph 173, p. 83. Amer. Chem. Soc., Washington, D. C., 1976. 3. Singer, B., and Grunberger, D., "Molecular Biology of Mutagens and Carcinogens," p. 65. Plenum, New York, 1983. 4. Loveless,A., Nature (London) 223, 206 (1969). 5. Lawley,P. D., in "ChemicalCarcinogensand DNA" (P. L. Grover, Ed.), Vol. 1, p. 1. CRC Press, Boca Raton, FI., 1979. 6. Koglin, E., S~quaris, J.-M., and Valenta, P., in "Surface Studies with Laser" (F. R. Aussenegg, A. Leitner, and M. E. Lippitsch, Eds.), p. 64. Springer-Verlag, Berlin, 1983. 7. Koglin,E., S~quafis,J.-M., Fritz, J. C., and Valenta, P., J. Mol. Struct. 114, 219 (1984). 8. Koglin,E., and S~quaris, J.-M., J. Phys. (Orsay, Ft.) 44, C10-487 (1983). 9. S~quaris, J.-M., Koglin, E., and Malfoy, B., FEBS Lett. 173, 95 (1984). 10. Balsinger, R. W., and Montgomery, J. A., J. Org. Chem. 25, 1573 (1960). 11. S~quaris, J.-M., Valenta, P., and Niirnberg, H. W., J. Electroanal. Chem. 122, 263 (1981).

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Journal of Colloid and Interface Science, Vol. 105, No. 2, June 1985