Chem. Res. Toxicol. 1990, 3, 559-565
559
Synthesis, Characterization, and Solution Properties of ras Sequences Modified by Arylamine Carcinogens at the First Base of Codon 61 M. Matilde Marques* Centro de Quimica Estrutural, Complex0 I , I.S.T., Au. Rouisco Pais, 1096 Lisboa Codex, Port uga 1
Frederick A. Beland* National Center for Toxicological Research, Jefferson, Arkansas 72079 Received J u n e 18, 1990
T h e complementary pentadecamers d(Y-TACTCTTCTTGACCT) (strand A) and d(5’AGGTCAAGAAGAGTA) (strand B), which span a portion of the mouse c-Ha-ras protooncogene centered around codon 61, were synthesized by using standard P-cyanoethyl phosphoramidite chemistry and characterized by sequence analysis. Strand A, containing a sole guanine at the position corresponding to the first base of codon 61, was modified with N-acetoxy-N-(trifluoroacetyl)-2-aminofluorene or its 4-aminobiphenyl analogue. In both cases only the corresponding N-(deoxyguanosin-8-y1)arylamine adduct was formed, as judged from HPLC and UV analyses conducted after enzymatic hydrolysis of the modified oligomers. Nonmodified and modified pentadecamers were annealed with strand B. Cooperative melting transitions were observed with all samples, thus indicating the formation of stable duplexes. Melting temperatures decreased in the order nonmodified duplex > 2-aminofluorene-modified duplex > 4-aminobiphenyl-modified duplex, which indicated destabilization of the helical structure upon incorporation of the adducts, with 4-aminobiphenyl having the greatest effect. Circular dichroism spectra of all duplexes were characteristic of an overall right-handed B-type conformation, with no major conformational differences being detected between the two arylamine-modified oligomers.
Introduction Aromatic amines and amides are a major class of chemical carcinogens that undergo metabolic activation to electrophilic derivatives capable of forming covalent adducts with DNA ( I ) . This adduct formation is regarded as a critical event in tumor induction since it may lead to mutations at crucial genomic sites (2). The major DNA adducts found after in vivo administration of arylamine (and arylamide) carcinogens are, in nearly all instances, arylamine C8-substituted deoxyguanosine adducts ( I ) . Despite this consistent pattern, different arylamines are known to induce distinct organ-specific toxicities in experimental animals ( I ) . In addition, arylamine-C8 adducts of deoxyguanosine exhibit different mutagenic efficiencies (3-7) and distributions in DNA (6-101, which appear to depend upon the nature of the aromatic moiety. In view of these observations, conformation-related effects may influence the biological properties of these DNA adducts. Most conformational studies with arylamine-DNA adducts have so far been conducted with 2-aminofluorene (AF) derivatives. Circular dichroism (CD) experiments have shown that chemical modification by 2-(acetylamino)fluorene (2-AAF) to yield N-(deoxyguanosin-8yl)-2-(acetylamino)fluorene(dG-C8-AAF) facilitates inversion of the alternating duplex poly(dG.dC) from normal right-handed B-DNA to left-handed Z-DNA (11,12). In this latter form, all guanines are in a syn conformation about the glycosydic bond, their C8 atoms are located externally, and no denaturation is observed in the modified 0893-228~ 190J 2103-0559$02.50/0
polymer. In contrast, in random nonalternating DNA sequences, where the Z-form is not favored (13),rotation of the modified guanines from the normal anti to the syn conformation will cause considerable distortion. This denaturation, which has been termed base displacement (14) or insertion-denaturation (15), results from the modified guanine being displaced from the right-handed helix while the AAF chromophore becomes inserted between adjacent bases. These models are supported by optical and NMR experiments (16-221, as well as by potential energy calculations with monomers, dimers, and short oligomers (20,23-26). Modification with the nonacetylated C8-substituted deoxyguanosine adduct, N (deoxyguanosin-8-yl)-2-aminofluorene ( d G - W A F ) , has also been shown to facilitate B Z transitions in poly(dGedC); however, higher modification levels and/or more drastic solvent conditions are required to achieve inversion (27). These and other experimental studies (17, 18,28-30) have implied that dG-C8-AF will cause less distortion of B-DNA than dG-C8-AAF, which is consistent with the higher in vivo persistence of the nonacetylated adduct (1). In agreement with these conclusions, theoretical models (23, 25, 31) have suggested the occurrence of a dynamic state with a variety of low-energy conformers including structures with anti-modified guanines and the A F chromophore in the major groove of a relatively undistorted B duplex. A recent combined NMR and computational study (32) has, nonetheless, presented evidence for an AF-modified syn guanine in a sequence with a guanine-
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560 Chem. Res. Toxicol., Vol. 3, No. 6, 1990
adenine base pair a t the modification site. Further calculations (33)indicated that similar structures, which place the arylamine fragment in the minor groove of the B-DNA double helix, may be energetically accessible and important during replication. Comparable studies with other arylamines are limited. CD data have shown that guanine modification with 4aminobiphenyl (ABP) to yield N-(deoxyguanosin-8-y1)-4aminobiphenyl (dG-C8-ABP) also facilitates a B Z transition in poly(dG.dC) (34). In addition, experimental results and potential energy calculations using the dimer d(CpG) have suggested the occurrence of similar minimum-energy conformers for AF- and ABP-modified species (35). However, subtle differences were detected, such as a higher stacking ability of the AF fragment, which might cause a better insertion of this arylamine in the double helix. Analysis of relatively short and selectively modified duplexes is one approach to evaluating conformation differences between structurally similar arylamine-DNA adducts. As a model, we have chosen the sequence of the mouse c-Ha-ras protooncogene from the last base of codon 59 to the second base of codon 64. A G T transversion of the first base of codon 61 has been observed in hepatomas from male B6C3F, mice treated with N-hydroxyAAF (36). This mutation is consistent with the formation of dG-C8-AF, which is essentially the only DNA adduct detected in the livers of these mice (37). We synthesized two complementary pentadecamers of which one, d(5'TACTCTTCTTGACCT), contained a single guanine a t the site of the reported in vivo mutation. This sequence was reacted with synthetic electrophilic derivatives of N-hydroxy-AF or N-hydroxy-ABP to give dG-C8-AF or dG-C8-ABP and then annealed with its complementary strand. Characterization and initial information on the solution properties of the nonmodified and modified duplexes are presented in this paper.
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Materials and Methods Synthesis and Characterization of Nonmodified Oligonucleotides. The pentadecamer d(5'-TACTCTTCTTGACCT) (strand A) and its complementary sequence d(5'-AGGTCAAGAAGAGTA) (strand B) were prepared on a 15-pmol scale with a Biosearch 8700 DNA synthesizer using standard 0-cyanoethyl phosphoramidite chemistry (38-40). All reagents for these syntheses were purchased from Biosearch Inc., San Rafael, CA. The 5'-tritylated products were released from the solid-phase support by ammonolysis a t 55 "C for 5 h and purified by reversed-phase high-pressure liquid chromatography (HPLC) with a Waters Associates system consisting of two 6000A pumps, a U6K injector, and a 660 solvent programmer. Peaks were monitored a t 260 nm with a Tracor 970 variable-wavelength detector. Separations were conducted with comparable results, using either a pBondapak CI8 (0.79 X 30 cm; Waters Associates, Milford, MA) or a Zorbax ODS (0.94 X 25 cm; Du Pont Instruments, Wilmington, DE) column. The solvent conditions were 1-6O% acetonitrile in 100 mM ammonium acetate (pH 5.7), with a nonlinear gradient (Waters no. 7) over 40 min a t a flow rate of 4 mL/min. Detritylation was achieved by treatment with 80% acetic acid a t room temperature for 2 h, and tritanol was removed by repeated extractions with water-saturated diethyl ether. The purity of the synthesized pentadecamers was confirmed by denaturing 20% polyacrylamide gel electrophoresis and by analytical reversed-phase HPLC, using either a pBondapak C18 (0.39 X 30 cm; Waters Associates) or a Vydac protein C4 (0.46 X 25 cm; Alltech Associates, Deerfield, IL) column. Each sample was quantified on the basis of absorbance data a t 260 nm. Extinction coefficients [ 128.6 and 168.3 L/(mmol.cm) for strands A and B, respectively] were calculated by the method of Cantor et al. ( 4 1 ) , using extinction coefficients of mononucleotides and dinucleoside monophosphates and assuming only nearest-neighbor
interactions. UV spectra of all samples were recorded with a Beckman DU-40 UV/vis spectrophotometer. The oligomers were subsequently characterized by DNA sequencing analysis, based on the chemical method of Maxam and (ICN P Gilbert (42). Following 5'-end labeling with [ Y - ~ ~ P I A T Biomedicals, Inc., Irvine, CA) and T4 polynucleotide kinase (Pharmacia/PL Biochemicals, Piscataway, NJ), chemical cleavage reactions were performed as described for short sequences (43, 44). After desalting and chain cleavage with 1 M piperidine a t 90 "C, electrophoresis was conducted a t 1500 V on a 20% polyacrylamide/8 M urea sequencing gel that had been prerun a t 25 mA for 30 min. Autoradiography was then performed a t -70 "C for 1-8 days with Kodak XAR-5 film. Modification of Strand A by 4-Aminobiphenyl or 2Aminofluorene. ABP modification of strand A was performed by incubation of the synthetic oligonucleotide with N-acetoxyN-(trifluoroacetyl)-4-amino[ 2,2'-3H]biphenyl. For this purpose, 4-nitr0[2,2'-~H] biphenyl (Chemsyn Science Laboratories, Lenexa, KS; 6.7 Ci/mmol) was diluted with unlabeled 4-nitrobiphenyl (Aldrich Chemical Co., Milwaukee, WI) to a final specific activity of 6.5 mCi/mmol, reduced to N-hydroxy-ABP by the method of Westra (45),and converted to N-acetoxy-N-(trifluoroacety1)-ABP by an adaptation of the method of Lee and King (46). In a typical modification, strand A (1.5 pmol) was dissolved in 400 pL of 10 mM sodium citrate (pH 6.0) and incubated for 1 h a t 37 "C with added in 45 pL 3.75 pmol of N-acetoxy-N-(trifluoroacety1)-ABP of ethanol. Three more addition-incubation steps were conducted, and then the reaction mixture was extracted with diethyl ether (8 X 1 volume) until no radioactivity was detected in the organic phase. The major reaction product was separated from the starting oligonucleotide by reversed-phase HPLC using a pBondapak CIa (0.79 X 30 cm) column and the solvent conditions used previously to purify the nonmodified oligonucleotides. The purity of the ABP-modified pentadecamer was confirmed with the analytical pBondapak Cl8 column, and the extinction coefficient was determined by absorbance and radioactivity measurements. AF modification of strand A followed a similar procedure. N-Hydro~y-2-amino[ring-~H]fluorene (Chemsyn; 58 mCi/mmol) was diluted with unlabeled hydroxylamine, which was obtained by reduction (45)of 2-nitrofluorene (Aldrich). The mixture was recrystallized from argon-purged methylene chlorideln-hexane (final specific activity 4.0 mCi/mmol) and converted to N-acetoxy-N-(trifl~oroacetyl)-2-amino[ring-~H]fluorene as described for ABP (46,47). Then, 2.5 pmol of the electrophilic ester in 50 pL of ethanol was added to 0.39 pmol of strand A in 250 pL of 10 mM sodium citrate (pH 6.0). The addition-incubation was repeated one additional time, and the AF-modified oligomer was isolated as described for the ABP-modified pentadecamer. Characterization of the Modified Pentadecamers. The identity of the ABP and AF adducts in the oligonucleotides was determined by enzymatic degradation. Each sample (15-20 pg) was dissolved in 100 pL of 5 mM Bis-Tris and 0.1 mM EDTA (pH 7.1). Following the addition of 2 pL of 1 M MgC1, and 10 pL of DNase I (Sigma Chemical Co., St. Louis, MO), a 3-h incubation was conducted a t 37 "C. Phosphodiesterase I (Sigma, 8 milliunits) and 0.33 units of alkaline phosphatase (Sigma) were then added, and the incubation was continued overnight. Each sample was centrifuged and the supernatant was analyzed by HPLC with the analytical pBondapak Cla reversed-phase column, using a 30-min nonlinear gradient (Waters no. 2) of 20-56% methanol a t a flow rate of 2 mL/min. Peaks were monitored a t 280 nm with a Hewlett-Packard 1040A diode array spectrophotometric detector. The 3H-labeled adducts were identified by comparison of their retention times and UV spectra with those of synthetic standards. Duplex Formation and Melting Experiments. Doublestranded oligodeoxynucleotides were prepared by mixing equimolar amounts of strand B with either nonmodified, ABP-modified, or AF-modified strand A. The samples were mixed in 10 mM sodium phosphate and 10 mM sodium chloride (pH 7.0), incubated a t 65 "C for 1.5 h, and then cooled to -20 "C for a t least 0.5 h. Melting curves were determined by monitoring the 260-nm absorbance with a temperature-controlled Cary 219 UV/vis spectrophotometer, while the temperature was raised from 14 to 60 "C a t a rate of -1 "C/min. Strand concentrations were estimated to be 3.3 pM for all the samples in these experiments.
Chem. Res. Toxicol., Vol. 3, No. 6, 1990 561
r a s Sequences Modified by Arylamines
b
A
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220
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400
Ahm)
I
20 Time (min)
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Figure 2. Reversed-phase HPLC of the reaction mixture, after
+
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1
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40
Time (min)
Figure 1. Reversed-phase HPLC of the reaction mixture, after extraction with diethyl ether, from modification of d(5'TACTCTTCTTGACCT) with N-acetoxy-N-(trifluoroacetyl)-2amino[rir~g-~H]fluorene. Solvent conditions were 1-60% acetonitrile in 100 mM ammonium acetate, pH 5.7, using a nonlinear gradient (Waters no. 7 ) and a pBondapak CI8analytical column. Peak a corresponds to the nonmodified pentadecamer and peak b to the AF-modified pentadecamer. The insets show the UV absorption spectra of both products, recorded in 10 mM sodium phosphate and 10 mM sodium chloride, p H 7.0.
Circular Dichroism Studies. CD spectra were measured a t r w m temperature, with a Jasco 5-500 spectropolarimeter, in 1-cm path length cuvettes. All samples were 11.7 pM in each strand. The instrument was calibrated with a 0.05% (w/v) solution of androsterone in dioxane, using a value of 192.4 millidegrees of ellipticity a t 304 nm. Results are reported as At, the difference in molar extinction coefficients between left-handed and righthanded circularly polarized light versus wavelength. These values were calculated from the observed ellipticities, using the nucleotide pair sample concentrations. All CD spectra were recorded under equilibrium conditions, usually 6-24 h after the solutions had been prepared.
Results and Discussion Synthesis and Characterization of Nonmodified and Arylamine-Modified Oligonucleotides. The pentadecamers d(Fj'-TACTCTTCTTGACCT) (strand A) and d(5'-AGGTCAAGAAGAGTA) (strand B) were synthesized, purified, and characterized by standard methods. The high specificity of electrophilic arylamine derivatives for reaction with the C8 position of guanine residues was taken advantage of in the selective modification of the single guanine in strand A. Figure 1 represents a typical chromatogram of the mixture from the reaction of strand
extraction with diethyl ether, from modification of d(5'TACTCTTCTTGACCT) with N-acetoxy-N-(trifluoroacetyl)-4amino[ 2,2'-3H]biphenyl, Elution conditions were as outlined in Figure 1. Peak a corresponds to the nonmodified pentadecamer and peak b to the ABP-modified pentadecamer. The inset shows the UV absorption spectrum of product b, recorded in 10 mM sodium phosphate and 10 mM sodium chloride, p H 7.0.
IZ5 n i
t 5
10
15
20 Time lmin)
25
30
35
40
Figure 3. Reversed-phase HPLC of the AF-modified pentadecamer after enzymatic hydrolysis. The histogram represents the radioactivity eluted from the column. Conditions were as outlined under Materials and Methods. Peak a corresponds to N-(deoxyguanosin-8-yl)-2-aminofluoreneand region b to products of spontaneous hydrolysis of product a. The inset shows the UV absorption spectrum of product a.
above 300 nm (Figure l),the type of extended conjugation expected from DNA containing dG-C8-AF adducts (47,48). The additional radioactivity was associated with incompletely extracted AF derivatives of low polarity: no other modified oligomers were detected. The typical yield of AF-modified strand A was -20%, and the molar extincA with N-acetoxy-N-(trifl~oroacetyl)-2-amino[ring-~H]-tion coefficient at 260 nm was 142.3 L/(mmol.cm). fluorene, obtained after diethyl ether extraction of most Reaction of strand A with N-acetoxy-N-(trifluorononbonded contaminants. Two major peaks were observed acetyl)-4-amin0[2,2'-~H]biphenyl was also highly specific. at 260 nm. The earlier eluting species contained no raAs shown in Figure 2, only two major peaks were detected dioactivity and was identified as the nonmodified pentaby HPLC at 260 nm. The earlier eluting species had decamer on the basis of its elution characteristics and UV chromatographic and UV absorbance characteristics spectrum (Figure 1). The second peak contained 40-50% identical with those of the starting oligonucleotide, while of the radioactivity eluting from the column. The UV the other major peak resulted from ABP modification. spectrum of this product exhibited an absorbance shoulder This latter peak accounted for 50% of the radioactivity
562 Chem. Res. Toxicol., Vol. 3, No. 6, 1990
Marques and Beland
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