Chem. Res. Toxicol. 1995, 8, 338-348
338
Multiple Fluorescence Lifetimes for Oligonucleotides Containing Single, Site-Specific Modifications at Guanine and Adenine Corresponding to Trans Addition of Exocyclic Amino Groups to (+)-(7R,8S,9S,lOR)and 7S,8R,9R,10S)-7,8-Dihydroxy-9,10-epoxy-7,8,9,10tetrahydrobenzo[a]pyrene (-)I(
Pierre R. LeBreton,*>*Chao-Ran Huang,' Harshica Fernando,' Barbara Zajc,t>s Mahesh K. Lakshman,:>"Jane M. Sayer,$ and Donald M. Jerina*lt Department of Chemistry, The University of Illinois at Chicago, Chicago, Illinois 60607-7061, and Laboratory of Bioorganic Chemistry, NIDDK, The National Institutes of Health, Bethesda, Maryland 20892 Received August 17, 1994's 5'-GGT CA C GAG-3'
Fluorescence decay profiles of four oligonucleotide duplexes, 3,.CCA GGG CTC.5, ((+I- and (-1trans-1) and ((+I- and (-)-trans-2), in which a n exocyclic amino group of deoxyadenosine (A*) or deoxyguanosine (G*)has been alkylated by trans opening at C-10 of the epoxide group of either the (+)-(R,S,S,R)-or (-1-(S,R,R,S)-enantiomer of (i)-7P,8adihydroxy-9a,10a-epoxy-7,8,9,l0-tetrahydrobenzo[alpyrene (BPDE in which the benzylic 7-hydroxy group and the epoxide oxygen are trans), exhibit more t h a n one fluorescence lifetime. Decay profiles of the oligomers, measured at 15 "C with excitation and emission wavelengths of 335 and 400 nm, respectively, have been analyzed using a triple-exponential decay law. Results for (+I- and (-)-trans-1 and -2 have been compared with results for the modified, singlestranded oligonucleotides ((+)- and (-)+-ans-SS-1, and (+)- and (-)-trans-SS-2) and for the cis and trans opened products formed on alkylation at the 6-amino group of 2'-deoxyadenosine 5'-phosphate by (+)-(R,S,S,R)-BPDE((+)-trans-and (+)&-A). The profiles of (+)-trans- and (+)-&-A are well represented by single-exponential decay laws with lifetimes of 86 and 110 i 3 ns, respectively. For the single- and double-stranded oligomer adducts, which exhibit a t least three fluorescence lifetimes, two of the lifetimes are short (0.5-14 & 1 ns) and one is long (35-59 4~3 ns). The fluorescence lifetimes and the amplitudes of the long-lived components in the decay profiles of the double-stranded oligomer adducts are generally smaller than those for the corresponding single-stranded adducts. The data provide evidence t h a t the doublestranded oligomer adducts exist a s multiple conformations. Previously reported NMR results suggest t h a t the short lifetime fluorescence components are due to major adduct conformations in which the pyrenyl group is intercalated ((+I- and (-)-trans-l) or lies in the minor groove ((+I- and (-)-trans-2). The observation of long lifetime fluorescence species for the doublestranded oligomers is consistent with the presence of minor conformations (-1-5%) in which the double-stranded oligomer either is locally denatured or is a mixture of locally denatured double-stranded conformations and equilibrium concentrations of single-stranded oligomers.
E::$
E':
Introduction The fluorescence lifetimes of high quantum yield metabolites of mutagenic and carcinogenic polycyclic aromatic hydrocarbons (PAH's)l are sensitive to binding interactions with DNA (1-8). This property makes information about fluorescence lifetimes of PAH metabolites, such as those derived from benzo[a]pyrene (BP), useful for examining the structures of mutagenic and carcinogenic DNA adducts. Much current evidence in' The University of Illinois at Chicago.
= The National Institutes of Health. 9 Present address: Department of Chemistry, University of Ljubl-
jana, ASkerEeva 5, 61000 Ljubljana, Slovenia. l 1 Present address: Chemsyn Science Laboratories, 13605 W. 96th Terr., Lenexa, KS 66215. Abstract published in Advance A C S Abstracts, February 1, 1995. Abbreviations: BP, benzo[alpyrene; BPDE, (&)-7/j,Ba-dihydroxy9a,10a-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene in which the benzylic 7-hydroxyl and epoxide groups are trans; ctDNA, calf thymus DNA; PAH, polycyclic aromatic hydrocarbon; tetraol, 7,8,9,lo-tetrahydroxy7.8,9,10-tetrahydro-BP. @
dicates that the exocyclic amino groups of deoxyguanosine (dG) and deoxyadenosine (dA)are the major sitesof covalent modification of DNA upon reaction either in vivo or in vitro with diol epoxide metabolites of PAH's (91,although minor adducts a t the exocyclic amino group of deoxycytidine and a t the ring N-7 of dG are also observed. The present investigation focuses on exocyclic purine amino group adducts derived by trans opening of the epoxide groups in the (+)-(R,S,S&)-and (-)-(S&&,S)enantiomers of (+t)-7P,8a-dihydroxy-9a,lOa-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene(BPDE with the benzylic 7-hydroxyl and epoxide groups trans). Conformations of covalent DNA adducts may be a n important factor in the expression of mutagenic and carcinogenic activities of PAH metabolites. This can occur through conformational effects on DNA interactions with polymerase and repair enzymes, with chromosomal proteins, and with regulating proteins. For example, adducts formed from cis and trans opened (+)-(R,S,SJ?)-and (-)-(S,RJ?,S)-BPDE exhibit different
0893-228x/95/2708-0338$09.00/00 1995 American Chemical Society
Fluorescence of BP Diol Epoxide Adducts
Chem. Res. Toxicol., Vol. 8, No. 3, 1995 339 R
OH [+]-(R,S,S,R)-BPDE
M o d i f i e d dA and dG i n [+]-trans oligomers
O'H [-l-(S,R,R,S)-BPDE
OH M o d i f i e d dA and dG I n [-]-trans o l i g o m e r s
Figure 1. Structures of the modified nucleosides in which a n exocyclic purine amino group has been alkylated by trans opening with either the (+j-(R,S,SP)-or (-)-(S,R,R,S)-enantiomers of (f~-7/3,8a-dihydroxy-9a,lOa-epoxy-7,8,9,l0-tetrahydrobenzo[alpyrene (BPDE in which the benzylic 7-hydroxyl and epoxide groups are trans).
influences on primer extension reactions of DNA polymerase I (10). Similar results have also been alsoobtained for elongation reactions catalyzed by T7 RNA polymerase (111. Differences in the environments which occur for different conformations of PAH metabolites, interacting with DNA either reversibly or covalently, give rise to different PAH metabolite fluorescence lifetimes (1-8). While the informational content of fluorescence lifetime data is not sufficient to provide detailed DNA adduct geometries, these data can provide qualitative features of adduct conformations. Specifically, the great sensitivity of fluorescence detection of high quantum yield adducts provides the opportunity to determine whether metabolites reside in one or in multiple conformations. The interpretation of results (3, 4, 7, 8, 12) from previous fluorescence lifetime measurements of adducts, formed in reactions of racemic BPDE ((&I-BPDE)with calf thymus DNA (ctDNA) (71, (+)-(R,S,S,R)- and (-1(S,R,R,S)-BPDEwith &DNA (4,121 and poly(dGdC).poly(dGdC) (41, and in reactions of (+)-(R,S,S,R)-BPDEwith poly(dAdT)poly(dAdT)(41, was hindered by photodecomposition of the adducts to give highly fluorescent 7,8,9,lO-tetrahydroxy-7,8,9,10-tetrahydro-BP (tetraol) and by the presence of multiple adduct types and sequences. Development of methods (13, 14) for the synthesis of oligonucleotides containing specific PAH adducts makes it possible to measure fluorescence lifetimes of samples in which all species have the same covalent structure. In the absence of contamination by tetraol, fluorescence lifetimes can provide evidence for the number of conformations of a given chemically homogeneous adduct. Here, we report results of fluorescence lifetime measurements of the modified oligonucleotides shown below: 5'-GGT CA*C GAG-3' 3'-CCA GG G CTC-5' (+)-and (-)-trans-I
5-CCA TCG*CTA CC-3' 3'-GGT AGC GAT GG-5 (+)-and (-)-trans-2
In these structures the central A* or G* represents a modified purine in which the exocyclic amino group is
covalently bonded to C-10 of BPDE by trans opening of the epoxide ring. As will be demonstrated later, photodecomposition of the adducted oligomers to tetraols proved not to be a problem under the present experimental conditions. The hydrocarbon portions of the trans opened epoxide adducts are shown in Figure 1. Oligomers with N-alkylated dA are designated as 1, and those with N-alkylated dG are designated a s 2 . Analysis of two-dimensional NMR data for (-)-trans-1 indicates a primary conformation which has the hydrocarbon intercalated (-80%) and a secondary conformation (-20%) in which the hydrocarbon may also be intercalated (15). Similar results have been obtained from a preliminary investigation of (+)-truns-1.2NMR has also been utilized to establish the solution conformations of the dG adducts in (+)- and (-)-trans-2 (16, 17). The pyrenyl group lies in the minor groove and points toward the 5'-end and the 3'-end of the modified strand in (+)and (-)-trunsd, respectively. In both of these latter cases, the NMR data provided no evidence for conformational heterogeneity (16, 17). Fluorescence intensities as a function of solvent polarity have been reported for (+)-truns-SS-2(18). Emission and excitation spectra have been reported for doublestranded oligomers containing 5'-d( CCT ATA G*AT ATC C) in which the guanine residue was modified with (+I(R,S,S,R)-and (-)-(S,R,R,S)-BPDE (19). For the doublestranded oligonucleotides, all bases of the unmodified strand, or all except one of the bases, were complementary with the modified strand (19). The results from fluorescence intensity measurements on the doublestranded oligomers were generally consistent with the NMR data for (+I- and (-)-truns-2 (16, 17). However, results from measurements of the influence of acrylamide on the emission intensities of duplexes formed from 5'd(CCT ATA G*AT ATC C ) suggest the occurrence of multiple conformations. H. J. C. Yeh, private communication.
340 Chem. Res. Toxicol., Vol. 8, No. 3, 1995
Materials and Methods Calf thymus DNA (ctDNA), purchased from Worthington (Freehold, NJ), contained less t h a n 1.2% protein, exhibited a n A26dAz80 ratio of 1.9, and had a hypochromicity of 35%. 7,8,9,lO-Tetrahydroxy-7,8,9,1O-tetrahydro-BP (tetraol) was obtained by hydrolyzing (&)-BPDE from Chemsyn Science Laboratories (Lenexa, KS). Acrylamide was purchased from Sigma (St.Louis, MO) and 9,10-dimethylanthracene from Aldrich (Milwaukee, WI). Experiments with tetraol and 9,lO-dimethylanthracene were carried out at concentrations in the range 10-6-10-7 M. Oligonucleotide concentrations are given in terms of the molarities of the intact single-stranded or double-stranded oligonucleotides, which were determined from U V absorption by use of the following concentrations (nmoUmL) corresponding to a n A260 of 1.0: trans-SS-l,9.3; trans-SS-2, 10.0; 5'-CTC GGGACC3' (the mismatched complementary strand for trans-l), 12.6; and 5'-GGT AGC GAT GG-3' (the complementary strand for trans2), 8.9.3 The concentration of ctDNA is reported in terms of PO4- molarity and was obtained from the relationship that 0.15 mM Po4- equals one A260 unit ( 5 , 20). Concentrations of the modified nucleotides (+)-trans-A and (+)&-A were estimated from A344 (a wavelength where the DNA bases have no absorbance) by use of an extinction coefficient of 4.76 x lo4 determined for (+ )-BPDE.
Synthesis of Modified Nucleotides and Oligonucleotides. Reaction of (+)-(R,S,SP)-BPDE with 2'-deoxyadenosine 5'-phosphate as described (21)provided (+)-trans- and (+)&-A i n which the exocyclic 6-amino group has opened the epoxide in a trans and cis fashion, respectively. Synthesis of the dA modified oligonucleotides (+)- and (-)-trans-SS-1was achieved
LeBreton et al. the oligomers were separated by HPLC on a Beckman Ultrasphere CIS column (5 pm, 10 x 150 mm) eluted a t 2.5 m u m i n with a linear gradient from 10% to 15% acetonitrile in 0.1 M ammonium carbonate buffer ( p H 7.5) over 20 min. These conditions resulted in products with homogeneous HPLC peaks and yields of 2.3 A260 (-)-trans-SS-2 at 11min and 2.2 A260 (+Itrans-SS-2 at 13 min derived from the (-)-(SJ,R,S)-and (+)(R,S,S$)-enantiomers of BPDE, respectively. Characteristic CD bands in the region of 300-350 nm, positive for the early and negative for the late eluting diastereomer, allowed assignment of absolute configuration (25, 26) of the early and late diastereomers a s 1OR and lOS, respectively. Thus, the early and late eluting diastereomers correspond to trans addition (with inversion of configuration a t C10) of the guanine base t o the (-)-7(S),8(R)-diol S(R),lO(S)-epoxideand (+)-7(R),8(S)-diol S(S),lO(R)-epoxide,respectively. The fully complementary duplexes, (-)- and (+)-trans-2,had T , 42 and 40 "C, respectively, at a total strand concentration of 0.01 mM. Fluorescence Measurements. Fluorescence measurements with nucleotide and oligomer adducts and with tetraol, a s well a s T , determinations, were carried out in 20 mM phosphate buffer (pH 7.0), adjusted to a n ionic strength of 100 mM with NaCl. Experiments with 9,lO-dimethylanthracene were carried out in 50% methanol in 10 mM Tris buffer (pH 7.3) containing 1.0 mM EDTA. Fluorescence quenching experiments with (+)-trans-A, (+I- and (-)-trans-SS-2, (-)-truns-2, (+)-trans-SS-l,9,lO-dimethylanthracene, tetraol, and tetraol in ctDNA were carried out in 0.125 M acrylamide. Solutions containing acrylamide were prepared by addition of 100 pL of 1.0 M acrylamide in the phosphate-NaCl buffer described to 700 pL of each sample employed in measurements of decay profiles without acrylamide. All fluorescence lifetimes and emission spectra were measured in air saturated solutions in 0.4 x 1.0 cm cells. Emission spectra were measured at 15 & 1 "C at excitation wavelengths of 335 and 355 nm with a PerkinElmer 650-10 fluorescence spectrometer. At excitation wavelengths of 385 and 410 nm, the resolution of the spectrometer was 14 nm (FWHM) under the conditions employed in these experiments. Fluorescence lifetimes were measured with a Photochemical Research Associates Model 2000 nanosecond fluorescence spectrometer. Lifetimes of tetraol and 9,lO-dimethylanthracene were measured at excitation wavelengths of 335 and 370 nm, and at emission wavelengths of 400 and 435 nm, respectively. Fluorescence lifetimes of the oligomer adducts were measured at excitation wavelengths of 335 and 355 nm, and a t a n emission wavelength of 400 nm. Collection of fluorescence lifetime data for each of the oligomer adducts required between 3 and 4 h. A previously described blank subtraction procedure was used to obtain reliable decay profiles (2, 5 , 6). The temperatures a t which the fluorescence decay profiles were determined are given in the figures. Fluorescence lifetime data were analyzed by a least-squares deconvolution method (27). The decay profiles of (+)-trans-and (+)&-A were fit with a single-exponential decay law. When a double-exponential decay law was employed, the x2 values obtained were not greatly improved over those obtained using a single-exponential decay law. Decay profiles of the single- and double-stranded oligonucleotide adducts were analyzed with a triple-exponential decay law:
through manual addition of a n appropriately blocked adduct phosphoramidite t o a support-bound tetramer prepared on a DNA synthesizer, followed by completion of the nonamer sequence on the synthesizer. The procedure is analogous to that reported earlier for the preparation of a nonanucleotide containing a phenanthrene diol epoxide (13). The single-stranded adducted oligomers were purified t o HPLC homogeneity on a Hamilton PRP-1 column (10 pm, 7 x 305 mm) using previously described conditions (13). The NMR structure of (-)-trum-l has been determined from a sample prepared in this manner (15). For the corresponding duplexes, (+I- and (-)-trans-1, the complementary base sequence selected has a dG mismatch opposite the adducted dA. This increased the transition temperature of the (+) duplex from 16 "C (with a complementary T) to 28 "C; both the (+) and (-) duplexes had T, 28 i 1 "C at a total strand concentration of 0.01 mM in the buffer used for the fluorescence measurements. The dG modified oligonucleotides, (+I- and (-)-truns-SS-2, have previously been synthesized by direct reaction of singlestranded, unmodified oligomer with diol epoxide (16,17). This method has also been applied to other guanine containing oligonucleotides (22). In the present investigation, (+I- and (-1trans-SS-2 were synthesized by postoligomerization modification (14, 2 3 ) . Except for the use of hexamethyldisiloxane which reacts with triethylammonium hydrogen fluoride that is formed in the reaction, the preparation of 5'-CCA TCI CTA CC-3', where I is 2-fluorodeoxyinosine, was as described (23). The fully blocked fluoro oligomer was left attached via its 3'-end to the controlled pore glass. Reaction of (d3-7P,8a,9a,-trihydroxy-10/3- I ( t ) = &[A, exp(-t/t,) +A, exp(-t/t,) amino-7,8,9,10-tetrahydrobenzo[a]pyrene(2.7 mg, 8.4 pmol), A, exp(-th,)l (1) obtained as described ( 2 4 ) ,and the bound fluoro oligomer (14 mg, 0.54 pmol), in 162 pL of 1 : l dimethylacetamidehexamethIn eq 1,Z ( t ) is the emission intensity at time t after excitation; yldisiloxane containing 2 pL of triethylamine at 55 "C for 5 days, l o is the intensity when t equals 0; TI, tz,and t3 are fluorescence followed by cleavage with ammonia, provided the desired lifetimes; and the amplitudes AI, Az, and A3 are constants. For oligomers. After removal of the dimethoxytrityl blocking group, a three-component system i n which the components have equal extinction coefficients, but different fluorescence lifetimes, the 3 Values of nmol of oligonucleotidelA260 were calculated by use of amplitudes AI, A2, and A3 are equal to the relative concentrathe program Oligo 4.0 (National Biosciences, Plymouth, MN) for the tions of the components. unmodified oligonucleotides. Estimates of the modified oligonucleotides In a calibration experiment, the fluorescence lifetime of 2,5were based on spectrophotometric titration (at 360 nm for trans-SS-1 diphenyloxazole (Fluka, Ronkonkoma, NY) was measured i n and 346 nm for trans-SS-2) with the complementary strands at undegassed spectrophotometric grade cyclohexane (Aldrich) a t temperatures below the T , of their duplexes.
+
Chem. Res. Toxicol., Vol. 8, No. 3, 1995 341
Fluorescence of BP Diol Epoxide Adducts
['I-GGT CA*C GAG C C A G G G CTC
600
15°C
-
[+I GGT C A T GAG CCA GG G CTC
, 1.
400
-
I+] trans 1 . .
i
W
1
I
!-
z -
[ + l C C A TCG* C T A CC
0
20
40
60
80
100
TIME (ns)
Figure 3. Comparison of fluorescence intensity versus time for (+)-trans-1 and t h e corresponding unmodified oligomer (0.013 mM) measured at excitation and emission wavelengths of 335 and 400 nm, respectively.
300
500
400
600
WAVELENGTH (nm) Figure 2. Fluorescence emission spectra of (+I-trans-1 and -2 (0.013 mM) measured in 20 mM phosphate buffer (pH 7.0, ionic strength 100 mM) a t 15 "C, utilizing excitation wavelengths of 335 and 355 nm.
20 "C, and at excitation and emission wavelengths of 300 and 440 nm, respectively. Results of the calibration experiment yielded a fluorescence lifetime of 1.16 n s with ax2value of 1.12. The lifetime obtained from the calibration experiment was similar to the value (1.28 ns) previously reported (28). In order to test for the presence of contaminating mononucleotide adducts, a supplementary experiment was carried out in which a sample of (-)-trans-2 was analyzed by HPLC, after the fluorescence lifetime measurements were completed. The analysis was carried out on the Hamilton PRP-1column described above with a linear gradient from 0% to 50% acetonitrile in 0.1 M ammonium carbonate buffer (pH 7.5) over 40 min a t a flow rate of 2.5 m u m i n . The elution was then continued for a n additional 60 min with 50% acetonitrile. The elution profile showed no evidence of modified mononucleotide.
Results Oligomer Adduct Fluorescence Emission Spectra. Fluorescence emission spectra of 0.013 M (+)trans-1 and -2, measured a t 335 and 355 nm excitation wavelengths and normalized so that the peak intensities are equal, are shown in Figure 2. Interestingly, the shapes of the spectra do not change when the excitation wavelength is changed. In contrast, shapes of previously reported emission spectra of adducts formed on reaction of (+)-(R,S,S,R)-BPDE with ctDNA (29, 30) depend on the excitation wavelength (335 or 355 nm). The emission spectrum of modified ctDNA, measured with a n excitation wavelength of 355 nm, has a broad shoulder between 460 and 520 nm (29, 30) that is decreased in intensity when the excitation wavelength is 335 nm. Similarly, the shape of the emission spectra of adducts formed from reaction of (+)-(R,S,S,R)-BPDE with poly(dG).poly(dC) measured a t excitation wavelengths of 340 and 345 nm is significantly different from the spectrum measured a t
355 nm (4,30,31). At 355 nm, the emission spectrum of the poly(dG).poly(dC) adducts has a broad maximum above 440 nm. This broad, long-wavelength emission reported in the polynucleotide adduct spectra has been attributed to pyrenyl excimers (30, 311, or possibly to pyrenyl-nucleotide base exciplexes (4). The excimer interpretation depends on the formation of adducts in which neighboring bases are modified (30). Since only one base per oligomer is modified in the present compounds, excimers are not possible. The observation that the shapes of oligonucleotide adduct emission spectra reported here are independent of the excitation wavelength supports the excimer interpretation of the emission spectra of the ctDNA and poly(dG).poly(dC)adducts. A comparison of the emission spectra indicates that the fluorescence intensities of (+)- and (-)-trans-1 adducts are larger than those of (+)- and ( - ) - t r a n s d . For example, a t excitation and emission wavelengths of 335 and 400 nm, respectively, the intensity of (+I-trans-1 is approximately 2 times greater than that of (+)-transd. This observation is consistent with previous results obtained when fluorescence detection was employed in the HPLC analysis of ribonucleoside adducts obtained from reactions of BPDE with polyribonucleotides (32).It is also consistent with results from measurements of the temperature dependence of fluorescence quantum yields for adducts obtained from reactions of BPDE with polydeoxyribonucleotides (33). At temperatures above 100 K, the adenine adducts exhibit stronger emission than the guanine adducts (32, 33). In addition to the spectra shown in Figure 2, emission spectra of (-)-trans-1 and -2 were also measured. The spectrum of (-1-trans-2 is similar to that of (+)-trans9. The spectrum of (-)-trans-1 is also similar to that of (+)trans-1. However, for (-1-trans-1 the intensities of the two bands observed in the spectrum are more nearly equal than f o r (+)-trans-1. Fluorescence Decay Profiles of Monomer and Oligomer Adducts. Fluorescence intensities of 0.013 mM (+)-trans-1 and of the corresponding unmodified duplex measured a t an emission wavelength of 400 nm as a function of time after a 335 nm excitation pulse are shown in Figure 3. Analysis of the data for (+)-trans-1 indicates the presence of high intensity, short-lived emission (containing at least two components, see below)
342 Chem. Res. Toxicol.,Vol. 8, No. 3, 1995
LeBreton et al. Table 1. Fluorescence Lifetimes of Oligonucleotides Modified w i t h (+)-(R,S,S&)-o r (-)-(S&,R,S)-Enantiomers of (~t)-7~,8a-Dihydroxy-9a,lOa-epoxy-7,8,9,10tetrahsdrobenzo[~Ipsrene~~~
15 o c
jtj t-1ton5
Vans A 12.111
I
(+
(+I 1-66nr
~ ~ - 0 9 5
I+] l,d”S ss-1 A1.051
~ ( - 3 5 ” s
A2-036
~ 2 - ? 3 n r
Ag-0‘3
x3-48nr
12.107
[+I-GGT C A T GAG CCA GG G CTC
[+Itrans 1 A2
-
-
r l - l O n r
0.32
Aj.002
7
1 0
PI
r3-37nr
2 2 I 1 13
1.1
GGT CA’C GAG CCA GG G CTC 1.1 Iran$ 1
Al-096
.rt-05ns
A\-003
~ 2 - 6 0 n r
A3.001
~ ~ - 5 7 n r
12.097
0
20
40
60
80
il
4.2“
cis A
(+].GGT CA’C GAG
Al.066
molecule 1-trans-A“
100
TIME (ns)
Figure 4. Fluorescence decay profiles for the trans and cis
opened adducts formed from reactions of (+)-(R,S,S,R)-BPDE with 2’-deoxyadenosine5’-phosphate ((-)-trans- and (+)-cis-A), for the single-strandedoligomer adduct (+)-trans-SS-1,and for the double-stranded adducts (+)- and (-)-trans-1. An instrument response profile is shown together with the decay profile for (-1trans-1. The response profile has a full width at half-maximum of 1.7 ns. Decay profiles were measured at excitation and emission wavelengths of 335 and 400 nm. Concentrations of the nucleotide adducts and of the oligomer adducts were 0.020 and 0.013 mM, respectively. Fluorescence lifetimes f r )and x2 values obtained from least-squaresfits of the data are given along with the decay profiles. For decay profiles which were fit with a tripleexponential decay law, amplitudes (AI, Az, and A d , associated with short lifetimes (rl and ra) and the long lifetime f r d , are also given. The scale indicating the log of the normalized intensity is shown on the lower left. and low intensity, long-lived emission. The intensities of both the short-lived and the long-lived emission from (+)-trans-1 are many times greater than the scattered intensity from the unmodified oligomer. The nucleotide adducts, (+)-trans- and (+)-cis-A,were examined to compare compounds in which a nucleotide base is covalently bound to the pyrene chromophore but the structured environment of a n oligonucleotide is absent. Fluorescence decay profiles for these nucleotide adducts consist almost entirely of a long-lived component with lifetimes of 110 and 86 i 3 ns, respectively (Figure 4). For (+I-trans-A, the fluorescence lifetime reported here is shorter than the value of 130 i. 5 ns reported for the nucleoside derived from (+)-trans-Ain air saturated solution (34). The conclusion that the fluorescence intensities of (+)and (-1-trans-1 adducts are larger than those of (+)- and (-)-trans-2 is consistent with the conclusion that the fluorescence lifetimes of (+)-trans- and (+)&-A are significantly larger than the fluorescence lifetime of the trans opened adduct formed by alkylation of the 2-amino
TZ
8.3”
i+)-cis-A’
i+)-trans-SS-l” 3.5 3.1” (+)-trans-ld 1.0 2.6’ i-)-trans-SS-ld 1.9 (-)-trans-ld 0.5 0.7’ ( + ) - t r a n ~ - S S - 22.0 ~ 1.7” ( + ) - t r ~ n s - 2 ~ 1.2 0.3’ (-j-trans-SS-2” 5.7 4.4” i - ) - t r ~ n s - 2 ~ 0.8 1.2’ 1.0’’
13 11”
4.0 4.8’ 14 6.0 10’ 9.5 7.7” 6.4 7.2’ 14 14” 9.0 9.7‘ 6.0”
r3
110 35” 86 48 34” 37 34’ 62 57 63’ 45 44” 35 34’ 51 56” 43 49’ 33”
A1
A2
A3
1.12 0.12” 0.79” 0.09’’ 1.02’’ 0.95 0.51 0.36 0.13 1.07 0.51’’ 0.36” 0.13” 1.11” 0.66 0.32 0.02 1.13 0.76‘ 0.21’ 0.03’ 1.08’ 0.65 0.21 0.14 1.18 0.96 0.03 0.01 0.97 0.97’ 0.02’ 0.01’ 1.17’ 0.38 0.50 0.12 1.07 0.47” 0.40” 0.13” 1.05” 0.76 0.19 0.05 1.11 0.92’ 0.06’ 0.02’ 1.15’ 0.48 0.27 0.25 1.16 0.56” 0.31” 0.13” 1.02” 0.84 0.13 0.03 1.01 0.71’ 0.25’ 0.04’ 1.09’ 0.75” 0.22” 0.03” 1.09’’
BPDE with the benzylic 7-hydroxyl and the epoxide oxygen groups trans. Fluorescence lifetimes (rl, r2, and ~ 3 in) ns obtained from decay profiles measured at 15 “C. For (+)-trans-Awith acrylamide and for the oligomer adducts the uncertainty in the lifetimes ( ~ 3 of) the long-lived components is 1 3 ns. The uncertainty in the lifetimes f r 1 and 521 of the shorter-lived components is k l ns. For the nucleotide adducts without acrylamide the uncertainty in the lifetime is 1 3 ns. All values obtained at an emission wavelength of 400 nm. Unprimed values obtained at an excitation wavelength of 335 nm without acrylamide. Singly primed values obtained at an excitation wavelength of 356 nm without acrylamide. Doubly primed values obtained at an excitation wavelength of 335 nm with 0.125 M acrylamide. Values obtained at a nucleotide concentration of 0.025 mM. Values obtained at an oligonucleotide concentration of 0.013 mM.
group of 2’-deoxyguanosine by (+)-(R,S,S&)-BPDE. In Nz purged water, this modified mononucleoside has a fluorescence lifetime of 1.6 ns (18). Transient excited state absorption measurements provide evidence that the small fluorescence lifetime of the adduct is associated with efficient hydrocarbon fluorescence quenching by guanine which arises from photoinduced electron transfer (35,36). In contrast to the results for (+)-trans- and (+)-cis-A in Figure 3, the dA modified single-stranded oligomer (+Itrans-SS-1 has two short-lived components (3.5 and 13 f 1 ns) in addition to a long-lived component (48 f 3 ns). Decay profiles of the dA modified double-stranded oligonucleotides, (+)- and (-)-trans-1 (at 15 “C, T , -28 “C), also exhibit two short-lived components and one longlived component. The lifetimes (Table 1)of (+)-trans-1 (1.0 k 1,4.0f 1,and 37 i 3 ns) are shorter than or equal to the corresponding lifetimes of (+)-trans-SS-1(3.4 f 1, 13 i 1, and 48 k 3 ns). Furthermore, the amplitude of the long-lived component in the decay profile of (+)trans-1 (0.02) is smaller than the amplitude of the longlived component in the decay profile of (+)-trans-SS-1 (0.13). Results from a comparison of the long and short lifetimes and the amplitudes of the long-lived component for (-)-trans1 vs (-)-trans-SS-1 (Figure 4 and Table 1) parallel the results for (+)-trans-1 vs (+)-trans-SS-1. The lifetime measurements reported here are for air saturated solutions. In degassed solutions, in which there is no oxygen quenching, the lifetimes are expected to be longer. Furthermore, the influence of oxygen quenching is greater on long-lived emission than on short-lived emission. For example, the fluorescence
Chem. Res. Toxicol., Vol. 8, No. 3, 1995 343
Fluorescence of BP Diol Epoxide Adducts
GGT CA'C GAG
[+I
xz
0
[-I trans 5 5 - 2
trans 5s-1
20
x
= 1.07
40
60
80
o
20
2 = 1.1s
40
60
ao
(ns) TIME (ns) and (-)-trans-SS-1measured at temperatures of 15 and 60 "C. The oligomer adduct
TIME
Figure 5. Fluorescence decay profiles of (+Iconcentrations were 0.013 mM, and the excitation and emission wavelengths were 335 and 400 nm.
lifetimes of anthracene a t 20 "C, measured with excitation and emission wavelengths of 300 and 405 nm, in undegassed and degassed cyclohexane, are 4.10 and 5.23 ns, respectively (28). The fluorescence lifetimes of tetraol in oxygen saturated and oxygen free solution are 130 and 200 f 4 ns (7). The time range employed in the present fluorescence lifetime measurements was 80 ns. For 10 of the 18 decay profiles in Table 1which exhibit multiple lifetimes, this time range was less than 2 times the lifetime of the longlived emission. This can give rise to reported lifetimes for long-lived emission that are too short and amplitudes that are too large. For (-1-trans-SS-1,for which the longlived emission was followed for only 1.3 lifetimes, a supplementary measurement was made to estimate the influence that changing the time range has on the amplitude and the lifetime of the long-lived emission. In the supplementary experiment, the time range was 160 ns. For the 160 ns time range, the values of t 3 (64 ns) and A3 (0.10) for the long-lived emission are similar to the values (62 ns and 0.14) obtained using the 80 ns time range. Results for the type 2 oligomers with dG adducts are given in Table 1 and Supplementary Figure S-1. Decay profiles for (-)-trans-2 measured a t concentrations of 3.3, 6.5, and 13.0 pM were nearly identical, indicating that oligonucleotide intermolecular interactions do not significantly influence the results of the present fluorescence lifetime measurements (Supplementary Figure S-1). The results for the type 2 oligomers are qualitatively similar to those for type 1 oligomers with dA adducts. For all of the type 2 adducts, like the type 1 adducts, the fluorescence decay profiles exhibit multiexponential behavior. Two components with short lifetimes (0.8-14 ns) and one component with a long lifetime (35-51 ns) are obtained upon analysis with a triple-exponential decay law (see Supplementary Material for residuals obtained from analysis of the fluorescence decay profiles, Figure S-2). For the double-stranded type 2 adducts, the lifetimes are
1.7-7.1 times shorter, and the amplitudes of the longlived components are 2.4-8.3 times smaller than corresponding values for the single-stranded adducts. Fluorescence Lifetimes of Double-Stranded Oligomer Adducts at Different Excitation Wavelengths. Results obtained for (+)- and (-)-trans-1 and -2 a t excitation wavelengths of 355 nm (singly primed numbers in Table 1) and 335 nm (unprimed numbers) are generally similar. The lifetimes of all but one of the short-lived components differ by less than 2 ns at the two excitation wavelengths. The exception, (-1-trans-1, has a lifetime which increases from 6 to 10 ns when the excitation wavelength is changed from 335 to 355 nm. For all four of the double-stranded oligomer adducts, the differences between the lifetimes of the long-lived components a t the two excitation wavelengths are less than 6 ns. The general similarity of the fluorescence lifetimes measured a t excitation wavelengths of 335 and 355 nm provides further evidence that, under the present experimental conditions, excimer emission is negligible, since larger differences in the decay profiles measured a t the two excitation wavelengths would have been expected ( 4 , 37) if excimer emission occurred. Temperature Dependence of Fluorescence Lifetimes of Single-Stranded Oligomer Adducts. Fluorescence lifetimes of the single-stranded oligomer adducts (+I- and (-)-trans-SS-1 measured a t 60 "C are smaller or equal, within experimental uncertainty, to lifetimes measured a t 15 "C (Figure 5). The general decrease in the fluorescence lifetimes of (+)- and (-)-trans-SS-1 that occurs as the temperature increases, is most likely associated with increases in the rates of nonradiative processes that accompany increased molecular motion a t higher temperatures. This conclusion is consistent with the view that the fluorescence lifetimes and quantum yields of nucleotide adducts formed from BP epoxides are determined primarily by dynamic quenching processes involving intramolecular collisions (4,7). Measurements of tetraol in buffer alone indicate that, for a n increase in
LeBreton et al.
344 Chem. Res. Toxicol., Vol. 8, No. 3, 1995
15 O C
15 O C
(A) Tetrol = 135 nr x 2 = 1.01
It]
T
(e)
A3 = 0 09 T 3
Tetrol + 0.25mM CtDNA T = 129 ns x 2 = 1.02
I-] trans SS.2 [Acrylamide] = o 125 M Ai-056 r l - 4 4 n s Ap=O31 r 2 - 1 4 n s As-013
[Acrylam~de]I 0 0 M Ai-048 T1-57nS
40
-
30
-
-
20-
10-
I
- -
Ap-027 12-14ns Ag 0 25 T 3 = 51 ns
x2
--
[.I frdns 2 (Acrylamide] 0 125 M A1 I 0.75 r 1 1 0 ns Ap-022 r 2 - 6 0 n s A3-003 r3-33nS
20-
. ....
10-
.. I.
2 2 = 109
00-
20
40
w
eo
iw
TIME (no)
Figure 6. Fluorescence decay profiles of 7,8,9,10-tetrahydroxy7,8,9,10-tetrahydro-BP (tetraol) in 20 mM phosphate buffer (ionic strength 100 mM) without ctDNA or acrylamide (A), with 0.25 mM ctDNA (B), with 0.125 M acrylamide (C), and with 0.125 M acrylamide and 0.25 mM ctDNA (D). The excitation and emission wavelengths were 335 and 400 nm. For all measurements, the pH was between 6.8 and 7.0.
temperature from 15 to 60 "C, the fluorescence lifetime decreases from 135 to 95 k 3 ns. Evidence against Photodecomposition. The time range employed in the present fluorescence lifetime measurements, 80 ns, is similar to the range employed in the earliest fluorescence lifetime measurements of adducts formed from reaction of (f)-BPDE, and of (+I(R,S,S,R)-and (-)-(S,R,R,S)-BPDE, with ctDNA (8,121. This fact, and the observation that the fluorescence lifetimes of the long-lived component in the oligomer adduct decay profiles are similar to values (38-45 ns) which were obtained from the early measurements on ctDNA, and which were later ascribed to tetraol formed upon photoinduced decomposition of the adducts, made it necessary to examine the possibility that tetraol contamination influenced the present results. In the present experiments, photochemical formation of tetraol was ruled out, since the contribution from the long-lived component did not increase between initial and subsequent irradiations of the same sample. For example, in three consecutive measurements of the fluorescence decay profile of (-)-trans-1, each requiring 3-4 h, values of 57, 57, and 52 ns and 0.016, 0.014, and 0.007 were obtained for the lifetime and the amplitude of the longlived component. Acrylamide Quenching Experiments. In order to test further for tetraol contamination, tetraol and oligomer adduct fluorescence lifetimes were measured in 0.125 M acrylamide (Figures 6 and 7 and Table 1). For all of the systems examined, the steady-state fluorescence
116
[-]CCA TCG' CTA CC GGTAGC GATGG
O O L
o
r3-56ns
x 2 = 102
(0) Tetrol + 0.25 mM clDNA + 0.125 M Acrylamide 7 = 12 nr x 2 1.12
35 ns
(-ICCA TCG' CTA CC
(C)
30
-
x 2 = 102
Tetrol + 0.125 mM Acrylamlde T I 8.8 nr 2 2 1.13 a0
frans A
IAcrylamide] = 0 125 M Ai-012 r1-42ns Ap=O79 r 2 - 8 4 n s
~
0
20
_
_
40
_
_
_
_
w
_
_
M
_
_
~ ~
~
~
loo
TIME (nr)
Figure 7. Fluorescence decay profiles of (+)-trans-A,(-)-transSS-2, and (-)-trans-2 in 0.125 M acrylamide, measured a t excitation and emission wavelengths of 335 and 400 nm. For (-)-trans-SS-2,a decay profile obtained without acrylamide is also shown. For the nucleotide and oligomer adducts, the concentrations without acrylamide were 0.020 and 0.013 mM, respectively. With acrylamide, the concentrations were 0.017 and 0.011 mM. In all samples, the pH was between 6.8 and 7.0.
intensities measured in 0.125 M acrylamide, a t the excitation and emission wavelengths a t which the fluorescence lifetimes were measured, are smaller than the intensities without acrylamide. For example, the steadystate intensity of (-)-trans-2 decreases 3-fold. All the decay profiles for the oligomers in acrylamide are well fit with a triple-exponential decay law, and the fluorescence lifetimes are smaller or equal, within experimental uncertainty, to lifetimes obtained without acrylamide. Acrylamide significantly shortens the fluorescence lifetime of tetraol (Figure 6), both in the presence and in the absence of DNA. All of the tetraol decay profiles are well represented by a single-exponential decay law. In the absence of acrylamide, fluorescence lifetimes obtained with and without ctDNA are nearly equal, with values of 129 and 135 i 3 ns, respectively. This observation is consistent with previously reported fluorescence lifetime data (1,2,6 , 3 8 )which indicate that, when BP metabolites and metabolite model compounds bind reversibly to DNA, the fluorescence quantum yields of the bound complexes are negligible compared to those of the free hydrocarbons. Thus, only emission from free tetraol contributes to the decay profiles of tetraol in the presence of ctDNA. In the presence of acrylamide, the tetraol fluorescence lifetimes, with and without ctDNA, are decreased to 12 and 8.8 f 1 ns, respectively. Similar results (not shown), were obtained for 9,lO-dimethylanthracene without DNA. Both with and without acrylamide, the decay profiles of 9,lO-dimethylanthracene
Fluorescence of BP Diol Epoxide Adducts follow a single-exponential decay law, and the lifetime obtained in 0.125 M acrylamide, 4.6 f 1 ns, is smaller than the lifetime, 14 f 1ns, obtained without acrylamide. Shorter fluorescence lifetimes and lower steady-state intensities obtained in the presence of acrylamide are due to dynamic quenching, where electronically excited tetraol undergoes radiationless transitions after collision with a n acrylamide molecule. Fluorescence decay profiles of (+)-trans-A,(-)-transSS-2, and (-)-trans-2 measured in 0.125 M acrylamide, as well as (-)-trans-SS-2 obtained without acrylamide, are shown in Figure 7. For (+)-trans-A,the decay profile obtained with acrylamide exhibits multiexponential behavior and is significantly different from the profile obtained without acrylamide (Figure 4). When a tripleexponential decay law is used to fit the decay profile of (+I-trans-A with acrylamide, the lifetimes (4.2 f 1, 8.3 f 1,and 35 f 3 ns) are all shorter than the lifetime (110 f 3 ns) of (+)-trans-A without acrylamide. A reliable interpretation of the difference between the shapes of the decay profiles for (+I-trans-A,obtained with and without acrylamide, will require further investigation of conformational influences on dynamic quenching and of the relative significance of dynamic versus static quenching mechanisms. For (-)-trans-SS-2and (-)-trans-2,the decay profiles measured in 0.125 M acrylamide resemble those measured without acrylamide. This observation and the finding that the steady-state intensity is quenched by acrylamide suggest that, for the oligomer adducts, dynamic quenching is accompanied by static quenching, where acrylamide reversibly binds to the oligomer adducts in the ground electronic state and forms a complex with negligible fluorescence quantum yield. However, like acrylamide quenching of (+)-trans-A,a full description of the quenching mechanism of the oligomer adduct fluorescence cannot be provided a t this time. The most important conclusion is that acrylamide has a smaller influence on the fluorescence lifetime of the long lived component in the decay profiles of the oligomer adducts than it has on the lifetime of tetraol, both with and without ctDNA. For (+)-trans-SS-1and (-)-trans-2 and (+I- and (-1-trans-SS-2 the lifetimes (34,33,44,and 51 f 3 ns, respectively) of the long-lived components obtained in 0.125 M acrylamide are only 1.4, 1.3, 1.02, and 1.1 times shorter than the lifetimes of the long-lived components obtained without acrylamide. In contrast, for tetraol with ctDNA, the lifetime obtained in 0.125 M acrylamide is more than 10 times shorter than that obtained without acrylamide. This marked difference in the effect of acrylamide on the lifetime of the long-lived component in the decay profiles of the oligomer adducts as compared to tetraol in the presence of ctDNA provides further evidence that the long-lived emission in the oligomer adduct decay profiles does not arise from tetraol contamination. The lack of tetraol contamination found in these experiments is similar to that recently reported in other fluorescence measurements of oligonucleotides modified with (+)-(R,S,S&)-and (-)-(S,R&,S)-BPDE (19).
Discussion The finding that the fluorescence decay profiles for the type 1 (trans-dA)and type 2 (trans-dG)oligomer adducts are qualitatively similar, although NMR results indicate that (+I- and (-)-trans1 contain a major intercalated
Chem. Res. Toxicol., Vol. 8, No. 3, 1995 345 conformation2 whereas (+I- and (-)-trans-2 contain adducts which lie in the minor groove with the plane of the aromatic ring system perpendicular to that of the paired bases (16, 17), demonstrates that fluorescence lifetime measurements cannot be used to identify specific types of adduct conformations. Furthermore, (+)- and (-)trans-2 exist in only a single conformation (or average of similar, rapidly-equilibrating conformations) detectable by NMR, whereas (+)- and (-)-trans-1 exhibit both major (-80%) and minor (-20%) conformations that equilibrate slowly on the NMR time scale. In contrast, all four oligomer duplexes give fluorescence decay profiles with a t least three components (Figures 3, 4, and S-1, and Table 1). Two possible explanations for the observed multiexponential decay profiles are the presence of a contribution from excimer emission or contamination of the samples by tetraol produced by photodecomposition. Contributions from eximers were considered negligible since emission spectra a t different excitation wavelengths (Figure 2 ) are nearly identical. Furthermore, fluorescence decay profiles measured a t different oligomer adduct concentrations (Supplementary Figure S-1) provide evidence that, a t the concentrations employed in the present experiments, the multiexponential decay profiles exhibited by the oligomer adducts arise from monomers. The possibility that the longest-lived, low-amplitude emission could result from photochemically produced tetraol was excluded, since the amplitude of this longlived component did not increase upon consecutive fluorescence lifetime measurements of the same sample. The dissimilarity in the quenching effect of acrylamide on tetraol in the presence of ctDNA (Figure 6 ) and on the adducted oligomers (Figure 7 and Table 1) further demonstrates that the long-lived emission is not due to tetraol contamination. Any interpretation of the present data must account for the multicomponent fluorescence decay profiles of the adducted oligomer duplexes and must reconcile the fluorescence data with NMR data on their solution structures. For all of the double-stranded oligomers (Table 11, the sum of the amplitudes of components with short fluorescence lifetimes ( < l o ns), as a fraction of the total amplitude, is 0.95 or greater. Thus, it is reasonable to assign both these short-lived components to the major conformation(s) observed by NMR. For (+)- and (-)trans-1, this conclusion is consistent with the expectation that intercalated adducts have low fluorescence quantum yields and short fluorescence lifetimes, since the fluorescence of BP metabolites is efficiently quenched by intercalation into DNA (1,2,6). The fact that oligomers with groove-bound dG adducts, (+I- and (-)-trans-2, also exhibit a t least two short-lifetime fluorescence components, but no evidence for structural heterogeneity by NMR, suggests that the conformations responsible for the short fluorescence lifetimes of these adducted oligomers are similar to each other andor rapidly interconverting on the NMR time scale. The assignment of the long-lived emission is more tenuous. Since the amplitudes of the long-lived components are small (15%), they are likely to correspond to conformations that are not detectable by NMR. It is unlikely that such conformations are similar to the principal conformations observed by NMR, since similar adduct geometries are not likely to give significantly different fluorescence lifetimes. Fluorescence lifetime measurements of reversibly bound complexes formed
346 Chem. Res. Toxicol., Vol. 8, No. 3, 1995
between BP metabolites and DNA support the conclusion that similar conformations give similar fluorescence lifetimes (1,2,6). The most reasonable explanation for the long-lived emission from (+)- and (-)-trans1 and -2 is that it arises from a small population of adducts with conformations significantly different from those that are observed by NMR and which exhibit short-lived emission. The high sensitivity of detection of the minor population of conformations results from their large fluorescence quantum yields, as indicated by their long fluorescence lifetimes. We speculate that the minor conformations responsible for the long-lived emission from (+I- and (-)-trans-1 and -2 have geometries in which the interaction between the adducts and the nucleotide bases is weaker than in the major intercalated or groove-bound conformations observed by NMR. This speculation is based on the finding (1,2 , 6) that strong reversible interactions between BP metabolites and nucleotide bases, such as those occurring in intercalated complexes, cause large reductions of the metabolite fluorescence quantum yields and lifetimes. The conclusion that the fluorescence lifetime decreases as the interaction between the nucleotide bases and the pyrene moiety increases is supported by the observations that the fluorescence lifetime of free tetraol(135 i= 3 ns) is longer than that of any of the adducts; that the lifetimes of (+)-trans- and (+)&-A (110 and 86 i 3 ns) are longer than those of the oligonucleotide adducts; and that lifetimes of the single-stranded oligomer adducts are generally longer than corresponding lifetimes of the double-stranded adducts. It is also supported by the finding that the amplitudes of contributions for long-lived emission, with fluorescence lifetimes greater than 15 ns, decrease as the environment of the chromophore becomes increasingly structured. For (+)-trans- and (+)&-A, virtually all of the emission is long-lived; for the singlestranded oligomer adducts, the amplitude of the longlived component is 0.25 or less; for the double-stranded adducts, the amplitude of the long-lived component is 0.05 or less. The speculation that the longer fluorescence lifetimes of the minor components of (+)- and (-)-trans-2 are associated with more exposed conformations is consistent with data from earlier fluorescence intensity measurements of other double-stranded oligomers containing dG residues modified with (+)-(B,S,S,R)and (-)-(S,RJl,S)BPDE (19). However, this speculation must be considered together with the finding that the fluorescence lifetime of the trans opened adduct formed by alkylation of the 2-amino group of 2'-deoxyguanosine by (+I(R,S,S,R)-BPDE is 1.6 ns when measured in aqueous medium (18). The small fluorescence lifetime of a mononucleoside adduct in which the DNA superstructure is minimized appears to provide evidence against an interpretation that relates long fluorescence lifetimes to conformations with weak base-hydrocarbon interactions. However, the short fluorescence lifetime of the deoxyguanosine-BPDE adduct may also rely on folded conformations in which there are strong reversible intramolecular hydrocarbon-base interactions within the adduct. The reversible binding of PAH's to nucleotide bases is strongly influenced by solvent polarity. For example, the ctDNA association constant of 9,lO-dimethylanthracene in water (-7 x lo3 M-')is reduced more than 12 times when methanol is added to a mole fraction of 0.31 (39). The conclusion that, for the oligonucleotide adducts examined here, a reduction in reversible hydrocarbon-
LeBreton et al. base interactions gives rise to an increase in fluorescence lifetimes is supported by the observation that when methanol is added to a mole fraction of 0.64, the fluorescence lifetime of the mononucleoside, deoxyguanosine-BPDE adduct, is more than 8 times larger than the lifetime in pure water (18). It is also interesting to consider the finding that the fluorescence decay profile of the single-stranded oligomer adducts, (+)- and (-)-trans-SS-1, which are completely denatured, exhibit multiple lifetimes, instead of longlived emission with a single lifetime. However, singlestranded DNA exhibits a wide range of geometries, in some of which there are strong local intra- and interstrand interactions. These interactions give rise to partially stacked regions, and even to local duplex formation, in which hydrocarbon quenching occurs. This has been observed for linear and for closed-circular single-stranded polymeric DNAs (1,40). It is likely that the distribution of conformations assumed by singlestranded oligomer adducts contains folded, stacked, and local duplex geometries which favor hydrocarbon quenching. What is important is that conformations which favor quenching occur with greater frequency in doublestranded oligomers than in single-stranded oligomers. Although the longer fluorescence lifetimes of the minor components of (+)- and (-)-trans-1 and -2 suggest that they arise from geometries that are less strongly associated with the nucleotide bases than the intercalated or groove bound conformations identified by NMR spectroscopy, fluorescence quenching experiments with acrylamide provide evidence that the pyrene chromophore in these minor species is less exposed than it is in free tetraol or in (+)-trans- and (+)&-A. Acrylamide has a greater influence on the decay profiles of tetraol, and of (+)-trans-and (+)-&-A, than on the decay profile of (-1trans-2. The relatively small influence of acrylamide on the lifetime of the long-lived component in the decay profile of (-)-trans-2 indicates that, even in this minor component, the adduct remains protected by the oligonucleotide structure. This is consistent with the observation that (+)-trans-SS-1 and (+I- and (-)-trans-SS-2 (Figure 7 and Table 1) are also much less sensitive to quenching by acrylamide than the monomeric nucleotide adducts or tetraol. Thus, even in single-stranded DNA, interactions with the nucleotide bases must provide significant protection of the adduct from dynamic quenching by acrylamide. In the decay profiles of the double-stranded oligomer adducts, the long-lived fluorescence might arise exclusively from a small fraction of single-stranded oligomer in equilibrium with the duplex. Evidence against this interpretation is provided by a comparison of the amplitudes for the long-lived components in the decay profiles of corresponding double-stranded and single-stranded oligomer adducts. For example, the amplitude of the long-lived components for (+)-trans-2 is 0.41 times as large as that for (+)-trans-SS-2. This indicates that if duplex dissociation is solely responsible for the long-lived emission, then significant dissociation (-40%) must occur. However, the temperature for the fluorescence measurements (15 "C) is 27 "C lower than the T,,, of (+Itrans-2 and lies well below the beginning of the sigmoid curve of absorbance vs temperature corresponding to the temperature dependent dissociation of the duplex. A reasonable interpretation is that the long-lived emission in the decay profiles of the double-stranded oligomer adducts arises in part from minor conformations
Fluorescence of BP Diol Epoxide Adducts in which local denaturation occurs a t the modification site. This interpretation is consistent with the observation that the fluorescence decay profiles of the singlestranded oligomer adducts, which are models for adducts in locally denatured DNA, show amplitudes for the longlived emission that are intermediate between those of the nucleotide adducts, (+)-trans- or (+)&-A, and those of the double-stranded oligomer adducts. It is also consistent with the observation that more-exposed conformations expected a t locally denatured sites are likely to give rise to longer-lived fluorescence when compared with structured conformations in which there is stronger interaction between the bases and the pyrene moiety.
Conclusions In summary, the main conclusions of this investigation are as follows: (1)Fluorescence decay profiles of oligonucleotide duplexes containing major intercalated (transdA) and groove-bound (trans-dG) adducts of BPDE are qualitatively similar. Unless data are collected on a larger number of oligomer adduct sequences, it is not likely that room temperature fluorescence lifetimes can be used for diagnosis of the gross conformation of these adducts. (2) Adduct duplexes whose NMR spectra are consistent with a single conformation as well as those that are conformationally heterogeneous by NMR both give fluorescence decay profiles with at least three components. When a triple-exponential decay law is used to analyze the decay profiles, two short-lived (I 10 ns) and one long-lived (35-57 ns) components are obtained. The two short-lived emissions account for 95% or greater of the total observed amplitude. Thus, it is likely that the species observed by NMR are responsible for the short-lived fluorescence. For the trans-dG adducts, whose NMR spectra indicate a single conformation, the two short lifetimes may arise from similar and/or rapidly equilibrating conformations that appear as a single species on the NMR time scale. (3)Long-lived emissions observed for the double-stranded adducts arise from minor conformations which, based on their relative amplitudes, make up 1-5% of the total population a t 15 "C and are thus insignificant contributors to the observed NMR spectra. Characteristics of these long-lived emissions are consistent with locally denatured doublestranded adduct conformations, or a mixture of locally denatured double-stranded adducts and equilibrium concentrations of single-stranded adducts. It is not clear whether minor adduct conformations with geometries that differ from the major intercalated and groove-bound conformations influence mechanisms associated with BP mutagenesis and carcinogenesis. However, the conclusion that the (+I- and (-1-trans-1 and -2 adducts examined here occur in multiple conformations may be significant in view of recent results which provide evidence that adduct conformational polymorphism influences the mutational frequency arising from (+)-(R,S,S,R)-BPDE adduct formation in the supF gene of the Escherichia coli plasmid pUB3 (41).
Acknowledgment. Support of this work by the American Cancer Society (Grant CN-371, the Petroleum Research Fund administered by the American Chemical Society (Grant 26499-AC), and the Blowitz-Ridgeway Foundation is gratefully acknowledged. H.F. would like to thank Searle for an Environmental Health and Safety Fellowship.
Chem. Res. Toxicol., Vol. 8, No. 3, 1995 347 Supplementary Material Available: Graphs of concentration dependence for fluorescence decay of (-)-trans-2 (Figure S-1) and of residuals from analysis of decay profiles (Figure S-2) (2 pages). Ordering information is given on any current masthead page.
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