Sequence-targeted photochemical modifications of nucleic acids by

Mar 1, 1990 - Preparation of hybrid "DNA cleaver-oligodeoxyribonulceotide" .... W. D. Wilson , Shaikh Mizan , Farial A. Taniuos , Shijie Yao , Gerald ...
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Bioconjugate Chem 1990, 1, 108-113

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Sequence-Targeted Photochemical Modifications of Nucleic Acids by Complementary Oligonucleotides Covalently Linked to Porphyrins Trung Le Doan,t D a n i d e Praseuth,t Loic Perrouault,t Marcel Chassignol,’ Nguyen T. Thuong,’ and Claude HBl&ne’yt Laboratoire de Biophysique, Museum National d’Histoire Naturelle, INSERM U201, CNRS UA 481, 43 rue Cuvier, 75005 Paris, France, and Centre de Biophysique Molbculaire, CNRS, 45071 Orleans Cedex 02, France. Received October 31, 1989

Porphyrins linked to oligonucleotides produce various types of photodamage on a complementary target DNA. The observed reactions include oxidation of guanine bases and cross-linking reactions of the oligonucleotide to its target sequence. Guanines located close to the porphyrin macrocycle were the most altered as compared to more remote guanines on the target sequence. No specific reaction was observed when the complexes were dissociated at temperatures above the melting temperature of the oligonucleotide-target hybrid. Both cross-linking and oxidation reactions accounted for ca. 60% modification of the target chains in the complex. Our results show that oligonucleotides covalently linked to porphyrins are efficient systems for inducing irreversible sequence-specific photodamage on a target DNA.

Synthetic oligonucleotides have been successfully used to control gene expression in various systems (For reviews see refs 1-3). However, in vivo applications of such oligonucleotides as antimessenger drugs face two main problems: (i) their poor penetration into cells and (ii) their susceptibility to nuclease degradation. Several strategies have been devised to cope with these problems. Oligonucleotides can be synthesized with a modified photodiester backbone so as to confer upon the whole molecule a better ability to cross cell membranes or to be protected from hydrolytic enzymes ( 4 , 5 ) . Another alternative is to synthesize oligonucleotides with the a-anomeric form of the nucleosides instead of the natural panomer (see ref 6 for a review). It was recently demonstrated that complexes of a messenger RNA and a complementary oligo-P-deoxynucleotide are specifically degraded by RNase H, a specific enzyme present in both prokaryotes and eukaryotes (7-10). These results constitute a strong basis for the understanding of the molecular mechanism of action of antimessenger oligodeoxynucleotides. Some of the oligonucleotide modifications (methylphosphonates, a-oligomers) that make them resistant to nucleases led to a loss of RNase H action on the mRNA-oligonucleotide hybrid (9, 10). This situation emphasized the need for developing oligonucleotides that could resist nuclease attack and induce local strand scission or chemical damage in their target. Such modified oligonucleotides could block enzymes involved in transcription or translation processes. For several years our group has been actively engaged in designing active oligonucleotides that can specifically bind to a nucleic acid sequence and subsequently generate, in a controlled process, irreversible damage on the target sequence. Specific irreversible damage can be successfully produced on target sequences by using complementary oligonucleotides synthesized with the natural (13) or synthetic ( a ) anomers of nucleotides and covalently Mus6um National d’Histoire Naturelle.

* Centre de Biophysique Molbculaire.

linked to metal complexes of EDTA, phenanthroline, and porphyrin derivatives in dark reactions (see refs 1 and 6 for reviews). Photosensitisers such as proflavin and azido derivatives (azidophenacyl and azidoproflavine) attached to natural and a-oligonucleotides were also shown to induce specific photochemical reactions on complementary sequences in single-stranded (11, 12) and doublestranded (13, 14) DNA, but the yield of these reactions was rather low. In this paper we present the results of a study of targeted photochemical reactions on DNA by complementary oligonucleotides linked to porphyrins. The reactions were found to be sequence-specific and the observed damage consisted mainly of cross-linking and oxidation reactions occurring predominantly on guanine bases of the target DNA. The yield of these reactions was much higher than that obtained with other photosensitizers. EXPERIMENTAL PROCEDURES A 27-mer oligonucleotidecontaining a stretch of adenines and whose sequence is

was used as a target for a series of porphyrin (P) substituted heptathymidylates

(Tp),T-O-CHz-CO-NH-(CH,),-NH-P 1 (Tp),T-O-CH,-CO-NH-(CH,),-NH-P 2 (TP),-(CyJrj-NH-P

P-NH-(CH,),-p(Tp),-(CH,),-Acr 4

(where P stands for methylpyrroporphyrin XXI (Aldrich), p for a phosphate group, and Acr for 2-methoxy-6chloro-9-aminoacridine). In all four compounds the carboxylic group of methylpyrroporphyrin XXI was attached to various linkers carrying an amine group to form an amide bond. In compounds 1 and 2, the linker was attached to the 3’-OH group of the 3’-terminal nucleo-

1043-1802/90/2901-0108$02.50/00 1990 American Chemical Society

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'0

O=P-O

'

-O -0 -O

O 0 I

-o-p=o

T

;

o I -o-p=o

: I I

I

I

0

I

"QT CH

CH3

methylpyrroporphyrin

x x

I

I H 2-met hoxy, 6-c hlo r o ,9-ami no ac r i d i n e

Figure 1. Schematic representation of compound 4 in which the porphyrin group is attached to the 5'-phosphate of the oligoheptathymidylate and the acridine derivative to the 3'-phosphate via a pentamethylene linker. For compound 3, the porphyrin was linked to the 3'-phosphate while in 1 and 2, it was linked to the 3'-OH of the oligonucleotide. For more details on linker structure, see text.

side, without any intervening phosphate group. In compounds 3 and 4 the linker was attached to the 3' (3) or 5' (4) phosphate group of the terminal nucleotide. The detailed structure of compound 4 is shown in Figure 1. Synthesis of the oligonucleotides was carried out on either an Applied Biosystems or a Pharmacia automatic synthesizer. The synthesis of oligothymidylate-dye conjugates has been previously described (15, 16). Purification of the oligonucleotides was done either by liquid chromatography or by gel electrophoresis. The 27-mer fragment was 5'-end labeled with T, polynucleotide kinase and T - [ ~ ~ P ] A T(Amersham). P A standard procedure consisted of successive additions in an Eppendorf tube of 5'-labeled 27-mer DNA fragment (10 nM), the oligo(dT),-porphyrin derivative (10 pM expressed as porphyrin concentration) in a final volume of 20 p L containing 10 mM phosphate buffer, pH 7.4, and NaCl, usually at 0.25 M final concentration. The mixture was kept in the dark at 0 "C for 1 h. The DNA solution was then transferred into a small glass tube, and irradiation was carried out a t 0 "C with the light of a high-pressure mercury lamp (200 W, OSRAM) filtered through a Pyrex glass plate (A > 300 nm). The incident light intensity a t the sample holder was measured with a Thermopile (Kipp and Zonen) to be approximately 180 mW/cm2. After irradiation, the sample solution was frozen and lyophilized. To characterize alkali-labile sites produced on the DNA, samples were submitted to piperidine treatment. The reacted DNA was dissolved with 50 pL of a 1 M piperidine solution and heated at 90 "C for 20 min followed by two cycles of washing with 50 pL of water and lyophilization. The reacted product was then redissolved in 10 pL of formamide-containing xylene cyano1 dye and loaded on a polyacrylamide gel (20% acrylamide containing bisacrylamide, 1:40 (M/M), 7 M urea). Autoradiograms were obtained by exposing the gel to Fuji (X-ray) films with an intensifying screen a t -80 "C overnight. Quantitative analysis of the reaction was carried out by excising the relevant bands from the gel and counting the corresponding radioactivity. RESULTS

1. Analysis of Photoproducts by Gel Electrophoresis. The autoradiogram presented in Figure 2 shows the photoproducts formed after irradiation of the com-

a 0

1

2

3

4

5

x -

*

XL1

-XL2 -XL3

T

G A v G T A A A A

* * .

A . A . A d

A

T G A

G

5'

~

Figure 2. Autoradiogram of photoproducts obtained by irradiating the 27-mer fragment (10 nM) in the presence of oligo(dT),-porphyrin (10 pM) in 10 mM sodium phosphate buffer, pH 7.4, containing 0.25 M NaCl. The left lane shows the (G A) sequence. Part of the 27-mer sequence is shown on the left. Lanes 1 and 2: compound 3, before (lane 2) and after piperidine treatment (lane 1). Lanes 3 and 4: compound 4, before (lane 4) and after piperidine treatment (lane 3). Lane 5: unirradiated 27-mer.

+

plex of two oligo(dT),-porphyrins (3, lanes 1and 2; and 4, lanes 3 and 4) with the target 27-mer whose (G + A) sequence is shown on the left side of the figure. At neutral pH, irradiation led to the production of new species (marked as XL,, XL,, and XL, in Figure 2) that migrated more slowly than the starting material (lanes 2 and 4). By analogy with what was previously observed with azido derivatives (11,12),these species can be ascribed to products formed by photo-cross-linking of the oligonucle-

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Le Doan et al.

t i m e (min)

Figure 3. Dependence on the irradiation duration of cleavage yields at G(8) (v),G(6) ( + I , and G(4) (M) after irradiation followed by piperidine treatment of the 27-mer in the presence of 3. Experimental conditions are as in Figure 2. The three lower curves represent the cleavage yields a t G(19) (upper), G(21) (middle), and G(23) (lower).

otide to the target matrix. The branched structure is expected to retard migration on the gels. The irradiated samples were then treated with 1 M piperidine at 90 “ C for 20 min in order to reveal alkalilabile sites (Figure 2, lanes 1 and 3). We observed that the cross-linked materials almost disappeared and that smaller fragments were produced that exhibited the same migration pattern as the fragments cleaved a t guanine bases produced by reaction with dimethyl sulfate in the Maxam-Gilbert sequencing reaction. Some cleavage was also observed at adenines and thymines in the vicinity of the A, terminus located close to the porphyrin ring, assuming that a Watson-Crick double helix is formed. Compounds 1 and 2 exhibited the same pattern of photoproducts but with much lower yield (see below). 2. Specificity of the Photochemical Reaction. In Figure 2, lanes 1and 2, the porphyrin moiety was attached at the 3’-end of the oligonucleotide and, as expected on the basis of an antiparallel orientation of the two strands, the main reactions occurred at G bases located on the 5’-side of the A, target sequence. The G bases on the opposite side (3’4de) were modified to a much lower extent. Conversely, when the porphyrin group was linked to the 5’-end of the oligonucleotide as in compound 4, the reaction occurred mostly at G bases of the 3’-side of the target (lane 3 of Figure 2). In a separate experiment with (dT),Acr we checked that the acridine dye (which was attached to the 3’-end of compound 4) was inactive under our experimental conditions (results not shown). The reaction specificity could be analyzed further by comparing the yield of cleavage at the different guanines as a function of their distance from the reacting porphyrin center. This is illustrated in Figure 3 for compound 3, where the yields of cleavage after piperidine treatment of the two groups of guanines located on either side of the target A, sequence are presented as a function of irradiation time. The reaction leveled off after 15 min of irradiation, corresponding to a total incident dose of -160 J/cmZ. This is very likely due to photodegradation of the dye at this irradiation dose. Such a photodegradation was observed by following the changes in absorption of the porphyrin ring under irradiation (data not shown). The much higher yield of cleavage at the 5’-guanines demonstrated the specificity reached with this system. As expected, the most modified G was G(8), the closest guanine to the photoactive group on the sequence. The cleavage yield decreased as one moved away from the reaction center on the 5’-side. For those guanines

0

5

20

10

30

t i m e (min)

Figure 4. Cleavage yields a t Gs on the 5‘-side of the target oligonucleotide [G(8) + G(6) G(4)] versus irradiation time after piperidine treatment of the reacted 27-mer in the presence of 1 (o), 2 (0),and 3 (A). Control experiment (A)consisted of irradiating the 27-mer in the absence of porphyrinoligonucleotide derivative. All other conditions are as in Figure 2.

+

located on the 3’-side, the reaction yield was very low, 2-3% going from G(19) to G(23). It should be noted that the cleavage yields at the latter Gs were close to background figures obtained when the matrix was irradiated in the absence of the porphyrin conjugates and subsequently treated by piperidine (1-2%) (see Figure 4). Similar results were observed on a 32-mer DNA fragment that did not contain the A, sequence. A nonspecific weak cleavage (1-270 per G base) was observed at all Gs in the presence of 3. 3. Influence of the Linker Length and Structure. The dramatic influence of the linker length and/or the chemical nature of the linker on the reaction efficiency is illustrated in Figure 4 for compounds 1-3. In compounds 1 and 2, the linker is directly attached to the 3’OH of the terminal thymidine while in 3 and 4 it is linked to the terminal phosphate group (see the Experimental Procedures and Figure 1 for a detailed structure of 4). Among the four compounds tested in this study, the most reactive compound was compound 3. Two amide bonds are present in the linker structure of 1 and 2 instead of one as in 3 and 4. The short length of the linker coupled with restricted flexibility of the amide bonds may account for the observed low efficiencies of reactions of compounds 1 and 2. Compound 4 exhibited an efficiency comparable to that of 2. The lower reactivity of 4 as compared to that of 3 may be due to the site of attachment of the porphyrin group on the oligonucleotide (5’-end instead of 3’-end in 3), even though the presence of an acridine on the 3‘-side provides a higher stability of the complex by intercalation of the acridine moiety within the duplex structure ( I ) . 4. Quantitative Analysis of the Photochemical Reaction with Compound 3. Irradiation of the complex under neutral pH conditions yielded three distinct cross-linked materials marked as XL,, XL,, and XL, in Figure 2. These cross-linked species and the material that migrated at the same position as the starting 27mer fragment were excised from the gel; the reacted DNA was extracted and purified by ethanol precipitation. The recovered samples were submitted to piperidine treatment. Results are presented in Table I and Figure 5. The values in Table I represent the radioactivity of each

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Table I. Quantitative Analysis of Photoproducts Formed after Irradiation (0 “C,15 min) of the Complex (27-mer (10 nM)-oligo(dT),-porphyrin 3 (10 pM)) in 10 mM Phosphate Buffer, pH 7.4, NaCl 0.25 M, before and after Piperidine Treatment

cross-linked 27-mer XLb = 37 non-cross-linked 27-mer 63”

XLI 3

XL, 21

XL, 13

After Piperidine Treatment

XL,“ XL2 XL3 27-mer (323) G(21) G(W A(10) to A(17) (38) G(6)

0.17 0.3 5.8 0.4 1.1 3 42 0.6 5.5 3.6 1.8d 0.05 0.3 0.4 0.7 0.04 0.4 0.3 0.4 0.07 0.3 0.5 1.25 0.17 0.45 0.6 6.0 0.4 2.9 1.1 2.9 0.3 1.4 1.1 (34) 2.4 0.2 0.8 1.1 C G(5’)/C G(3’)= 3.9 5.6 5.1 2.8 a All values indicate the percentage of radioactivity in the corresponding bands extracted from the gel. X L cross-linked materials (see Figures 2 and 5). “ Remaining cross-linked material after piperidine treatment. Corrected values taking into account cleavage at G and A bases induced by piperidine treatment of the irradiated 27-mer in the absence of the oligonucleotide derivative. e G(5’) = G(8) + G(6) + G(4) and G(3’) = G(19) + G(21) + G(23). 1

2

3 4

-XLl -XL2 -XL3

G 23 G 21 G 18

G8

G6

G4

Figure 5. Autoradiogram of photoreacted 27-mer in the presence of 3 under the experimental conditions indicated in Figure 2. The material migrating as the 27-mer band (lane 1)and the cross-linked products XL, (lane 4), XL, (lane 3), and XL, (lane 2) were extracted from the gel and treated with piperidine before loading on the gel. The radioactivity in each lane reflects the yield of each photoproduct as detailed in Table I.

band as a percentage of the total radioactivity in the lane. The non-cross-linked material migrating as intact 27mer yielded cleavage a t guanines after piperidine treatment (Figure 5, lane 1,and first column of Table I). The data in Table I show that 25% of what migrated as the intact 27-mer in fact contained photooxidized guanines that were cleaved under alkaline conditions. Under our experimental conditions, the yield of crosslinked products accounted for 37% of the original 27-

I 0

0.2 5

0.5

.

1.0

NaCl ( M )

Figure 6. Influence of ionic concentration on the photochemical reaction of 3 with the 27-mer fragment after 15-min irradiation. Cleavage yield after piperidine treatment of the three Gs on the 5’-side (upper curve) and of the three Gs on the 3’side (lower curve). NaCl (v,v), NaClO,) (+, O ) , in 10 mM sodium phosphate buffer pH 7.4.

mer. After piperidine treatment, there was about onethird of each of the cross-linked product left unmodified. The other two-thirds were converted into a product migrating as the starting material (27-mer) and shorter fragments cleaved predominantly at guanines (Figure 5, lanes 2-4). This result shows that piperidine treatment of the photo-cross-linked material led to cleavage of the 27-mer a t photooxidized bases and/or bases involved in the cross-linking reactions (mostly guanines) and to cleavage of the covalent bond formed between bases and porphyrin, thereby releasing the cross-linked oligonucleotide from the 27-mer. In the latter case the resulting product, which migrates as an intact 27-mer, might contain altered bases that are not alkali-labile sites. The specificity of the photochemical reaction is evidenced by comparing the yields of cleavage of the three Gs on the 5’-side to those located on the other side (last row of Table I). The results show that the probability of photochemical reaction is 3-5 times higher a t the Gs located in the vicinity of the photosensitizer (5’-side) as compared to those located on the opposite side of the target sequence. When the mixture of 3 and the target was irradiated at a temperature (30 “C) a t which the complex was dissociated, the ratio of cleavage yields a t Gs on the 5’- and 3’-side was reduced to about 0.8. Assuming that cleavage of Gs on the 3’-side [G(19), G(21), and G(23)] represent the oxidative reaction due to diffusing species (see the Discussion), there results showed that in the cross-linked materials (37% of the starting matrix), ca. 20% contained oxidized Gs in their structure. 5. Influence of Ionic Concentration. The stability of complementary DNA duplexes is known to increase when the ionic concentration increases as a result of a reduction in the repulsive interactions between the two negatively charged phosphodiester backbones.’ The dependence of the photosensitized reaction with ionic concentration is shown in Figure 6. It can be seen that the reaction yield of G modification on the 5’-side increased when salt concentration increased from 0.01 to 0.15 M and then remained constant up to 1.0 M NaCl (or NaC10,). The yield of cleavage a t the three Gs located on the 3’-side of the matrix remained constant over the whole concentration range of Na salts. This result demonstrates that the specific photosensitized reaction on the 5’-side is strongly dependent on the formation and stability of the oligonucleotide-target hybrid and the reactions a t guanines on the 3’-side are not due to the formation of a triple helix involving two oligo(dT)s bound to the oli-

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go(dA) sequence of the 27-mer. Such a triple helix would have been favored at high ionic concentration (14). 6. Influence of the Reaction Temperature. The temperature dependence of the photochemical reaction was studied with compound 4. In this compound the porphyrin group is attached to the 5'-end of the oligonucleotide and the acridine derivative to the 3'-end. Therefore the specific reaction occurs a t G(19). The reaction yield as measured by the cleavage at G(19) after piperidine treatment decreased when the temperature increased with a half-transition at -18 "C (results not shown). Photocross-linking before piperidine treatment was abolished at high temperature and the half-transition also occurred at 18 "C. In contrast, the nonspecific reactions at Gs on the 5'-side increased with temperature. In a previous work we studied the cleavage of a poly(dA) matrix in the dark by iron complexes of 3 and 4 (15). Melting temperatures of 17 "C and 32 "C were observed for the metal complexes of 3 and 4, respectively. These temperatures are certainly higher than those expected for the nonmetalated porphyrin due to the increased interaction provided by the positive charge brought by the Fe ion and more importantly by the effect of cooperative binding on the homopolynucleotide matrix. DISCUSSION

This study shows that specific photochemical reactions can be targeted to specific DNA sequences by irradiating the complex formed by the target sequence with a complementary oligonucleotide tethered to a porphyrin group a t the 3'- or Y-end. These reactions involve bound porphyrin-oligonucleotide conjugates as reaction only occurred when the two molecules were hybridized, Le., at high salt concentration and at temperatures lower than the melting temperature of the complex. Moreover, it was shown that the main reactions occurred on the side of the target sequence expected when the substrate and the porphyrin-oligonucleotide conjugate form a double helix with antiparallel orientation of the two strands. Both oxidation and cross-linking reactions were observed. Quantitative analysis of the photochemical reactions showed that under our experimental conditions, total damage, including oxidation and cross-linked products, accounted for ca. 60% of the target DNA for the most active derivative 3. Porphyrins strongly absorb visible light and the produced excited states can react with various substrates including nucleic acid bases ( I 7). When a porphyrin group is brought in close vicinity of a target DNA two types of photochemical events can be envisaged: (i) A direct reaction of the excited porphyrin (in its singlet or triplet state) with the substrate, e.g., an electron-transfer reaction from a guanine base yielding a G radical cation. (ii) The porphyrin excited states can react with oxygen, producing singlet oxygen '0, or the superoxide anion O,*-. The latter has been found to be rather inactive toward nucleic acid bases while '0, reacts readily with guanine and to a lesser extent with thymine bases (18,19). The observed alkali-labile sites at guanine bases in the target sequence could result from singlet-oxygen attack but also from decomposition of some peroxyl form of the guanine radical cation. The fluorescence of some cationic porphyrins has been found to be quenched by polyd(G-C) and calf-thymus DNA ( 2 0 ) , and this quenching has been ascribed to an electron-transfer reaction in the excited state. Photoreduction of hematoporphyrin has been observed in the presence of reductants such as ascorbate, pyrogallol, and catechol (21). Berg et al. (22) also proposed an electron-transfer reaction between thiopy-

Le Doan et ai.

ronine and guanine base, resulting in the formation of the radical cation Go+. The production of such radicals implies a close contact between donor and acceptor molecules. The photo-cross-linking reaction could result from a very localized reaction between radical species, in contrast to oxidation reactions involving diffusing species such as singlet oxygen. A mean diffusion pathway of singlet oxygen of 250 nm has been calculated from the diffusion constant (2.5 X lo-' m2 s-') and the lifetime of singlet oxygen in water (4 ps at 20 "C). The alkali-induced cleavage reactions at guanine residues do not occur randomly. This is shown after piperidine treatment of the band migrating as the intact 27-mer (Figure 5, lane 1). Cleavage occurs predominantly on the 5'-side of the target sequence, indicating that modified guanines have been mostly produced in the immediate vicinity of the porphyrin ring. Porphyrins have been shown to produce singlet oxygen as a result of energy transfer from the triplet state (23, 24). Guanine bases located in the immediate vicinity of the porphyrin ring should react rapidly with singlet oxygen. A large excess of oligonucleotideporphyrin conjugate over target DNA was used in most experiments. Singlet oxygen produced by unbound molecules is expected to react with guanine bases in a nonselective way. This is what was observed when the oligonucleotide-target hybrid was dissociated at temperatures above the melting temperature. All guanine bases were photooxidized both on the 5'- and the 3'4des of the A, sequence although the reaction yield was lower than with the bound oligonucleotide. In contrast, the photo-cross-linking reaction leading to slowly migrating species on neutral polyacrylamide gels was abolished when the complex was dissociated. In summary, porphyrinlinked oligonucleotide lead to three types of reactions. Photo-cross-linking of porphyrin with nucleic acid bases and local photooxidation of bases by singlet oxygen are sequence-specific and occur only when the oligonucleotide is bound to its target sequence. Some further nonsequence-specific photooxidation of bases occurs mainly at guanines, due to singlet oxygen produced by unbound oligonucleotides and, possibly, by singlet oxygen generated from bound oligonucleotide and diffusing away from the porphyrin. Literature data indicate that photochemicall modified Gs are effective blocking sites for various enzymes. Guanine oxidation mediated by singlet oxygen appears to generate efficient arrest sites for Escherichia coli DNA polymerase I (25, 26). Inhibition of DNA-dependent RNA synthesis by porphyrins has also been observed (27). Several photosensitizers have been shown to produce photodamage similar to those observed in our system. In a previous work we have shown that, under irradiation, proflavine induced both cross-linking and oxidation reactions occurring mainly at G bases (13). Piette and Moore (28) showed that damage photoinduced by proflavine blocked DNA polymerases on the reacted matrix. In the phenothiazine series, the genotoxic properties of chlorpromazin have been correlated with the ability to add covalently on to guanine bases of DNA (29). In model studies, Ciulla et al. (30) have isolated a photoadduct resulting from the coupling between the C(8) position of the deoxyguanosine and the C(2) position of the phenothiazine ring. The unreacted DNA was probed with DNA polymerase and the enzyme was found to stop one nucleotide before every guanine residue (29). Recently it has been reported that a specific photo-crosslinking reaction could be achieved with an oligonucleotide linked to a photoactivatable group, 4'-(aminoalkyl)4,5',8-trimethylpsoralen (31). Photoadducts of psoralens with thymine bases have been demonstrated to be

Sequence-Targeted Photochemical Modifications

efficient arrest sites for various enzymes including E. coli and T, DNA polymerases and reverse transcriptases (32, 33). In conclusion, site-directed photodamage produced by porphyrins or other sensitizers coupled to oligonucleotides appears to provide a promising system for the selective inhibition of gene expression. Porphyrins can be covalently attached to nuclease-resistant oligonucleotides. This should make these oligonucleotidesporphyrin conjugates suitable for in vivo applications. Work is in progress in our laboratory on selective control of gene expression a t replication, transcription, and translation levels using these new photoactive oligonucleotides. ACKNOWLEDGMENT

This work was supported in part by Rh8ne-PoulencSantB, the Ligue Nationale Francaise contre le Cancer, and the Fondation pour la Recherche MBdicale. We wish to thank Drs. J. Igolen and M. C. Gouyette, Institut Pasteur, Paris, for a gift of a sample of the 27-mer, and J. M. Kelly for helpful discussions. LITERATURE CITED (1) HBlBne, C. (1987) In DNA-Ligand Interactions (W. Guschlbauer and W. Saenger, Eds.) pp 127-140, Plenum Publishing Corp., New York. (2) Stein, C. A., and Cohen, J. S. (1988) Cancer Res. 48, 26592668. (3) ToulmB, J. J., and HBlBne, C. (1988) Gene 72, 51-58. (4) Miller, P. S., and Tso, P. 0. P. (1987) Anticancer Drug Des. 2, 117-128. ( 5 ) Matsukura, M., Shinokuza, K., Zon, G., Mitsuya, H., Reitz, M., Cohen, J. S., and Broder, S. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 7706-7710. (6) HBlBne, C., and Thuong, N. T. (1988) In Nucleic Acids and Molecular Biology, (F. Eckstein and D. M. J. Lilley, Eds.) Vol 2, pp 105-123, Springer Verlag, New York. (7) Minshull, J., and Hunt, T. (1986) Nucleic Acids Res. 14, 6433-6451. (8) Cazenave, C., Loreau, N., Thuong, N. T., ToulmB, J. J., and HBlBne, C. (1987) Nucleic Acids Res. 15, 4717-4736. (9) Gagnor, C., Bertrand, J. R., Thenet, S., Lemaitre, M., Morvan, F., Rayner, B., Malvy, C., Lebleu, B., Imbach, J. L., and Paoletti, C. (1987) Nucleic Acids Res. 15, 10419-10436. (10) Cazenave, C., Stein, C. A., Loreau, N., Thuong, N. T., Neckers, L. M., Subasinghe, C., HBlBne, C., Cohen, J. s.,and ToulmB, J. J. (1989) Nucleic Acids Res. 17, 4255-4273. (11) Praseuth, D., Chassignol, M., Takusugi, M., Le Doan, T.,

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Registry No. 1, 114436-49-6; 2, 112761-79-2; 3, 112726-406; 4, 114436-51-0; 27-mer oligonucleotide, 112603-07-3; G, 7340-5; 0 2 , 7782-44-7.