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TECHNICAL NOTES Detection of Psoralen Cross-Link Sites in DNA Modified by Psoralen-Conjugated Oligodeoxyribonucleoside Methylphosphonates Joanne M. Kean and Paul S. Miller* Department of Biochemistry, School of Hygiene and Public Health, The Johns Hopkins University, 615 North Wolfe Street, Baltimore, Maryland 21205. Received September 14, 1992
Oligodeoxyribonucleoside methylphosphonates conjugated with derivatives of psoralen cross-link with complementary single-stranded RNA and DNA and with duplex DNA targets when irradiated with long wavelength (365 nm) ultraviolet light. The position of cross-linking between pyranone-side adducts of psoralen-conjugated oligonucleosidemethylphosphonates and DNA can be easily detected by treating the photoadduct with 1M aqueous piperidine at 90 "C for 30 min, followed by analysis on a polyacrylamide gel run under denaturing conditions. This treatment results in hydrolysis of the methylphosphonate linkages and cleavage of the phosphodiester backbone at the cross-link site. Multiple cross-linking sites were detected with a single-stranded DNA target which contains four contiguous T residues. This may result from looping out of one or two of the T residues.
INTRODUCTION
Psoralen conjugates of oligodeoxyribonucleotides or oligonucleosidemethylphosphonates, when hybridized to a complementary nucleic acid, can be triggered to form a photoadduct upon irradiation with long-wavelength ultraviolet light. Various types of psoralen-conjugated oligomers have been prepared and shown to cross-link in vitro with both single-stranded RNA and DNA and with duplex DNA targets (1-1 7). In addition, psoralen-conjugated oligomershave displayed highly selectiveantisense and antigene activity in a number of biochemical and biological experiments (15, 18-22). Psoralen cross-linking sites in RNA can be located using a combination of selective enzymatic and chemicalcleavage reactions followed by 2-dimensional gel electrophoresis (23,24)or by using an oligodeoxyribonucleotide primer and reverse transcriptase (12,25). A method employing borohydride and aniline to cleave psoralen-thymidine photoadducts formed as a result of cross-linking psoralenconjugated oligodeoxyribonucleotides to single-stranded DNA has been described recently (16). In this Note we describe a simple procedure for locating the site of crosslinking between psoralen-conjugated oligodeoxyribonucleoside methylphosphonates and DNA targets which employs piperidine-mediated cleavage of the phosphodiester backbone at the cross-link site. EXPERIMENTAL PROCEDURES
Reagent-grade piperidine was distilled under an argon atmosphere and stored under argon at -80 "C. Solutions containing 1.0 or 1.25 M distilled piperidine in deionized water were freshly prepared just prior to use. Protected deoxyribonucleotide 3'-[@-cyanoethyl N,N-diisopropylphosphoramiditesl were purchased from Glen Research Inc. and the protected deoxyribonucleoside 3'- [methyl N,N-diisopropylphosphonamiditeslwere a gift from JBL Scientific, San Luis Obispo, CA. Polyacrylamide gels were run using a gel buffer which contained 0.045 M Tris, 0.045 1043-1802/93/2904-0104$04.00/0
M boric acid, and 0.05 mM ethylenediamine tetracetate buffered at pH 8.0. All reagents used were reagent-grade or better. The sequences of the oligodeoxyribonucleotides and psoralen-derivatized oligonucleoside methylphosphonates are shown in Figure 1. Syntheses of Oligodeoxyribonucleotides and Oligodeoxyribonucleoside Methylphosphonates. The oligodeoxyribonucleotide targets I and I11 were prepared on controlled pore glass supports in a Biosearch Model 8700 DNA synthesizer using standard phosphoramidite chemistry (26). The 5'4erminal dimethoxytrityl group of the protected oligomer was removed by the synthesizer, and the oligomer was then deprotected and removed from the support by heating with concentrated ammonium hydroxide in pyridine (1:l v/v) at 55 "C for 5 h. The oligomers were phosphorylated using polynucleotide kinase and [y3*P1ATP ( 2 3 , and the phosphorylated oligomers were purified by gel electrophoresis on a 30 cm X 40 cm, 20% polyacrylamide gel which contained 7 M urea run at 1800 V for 5 h. The oligomers were extracted from the gel at 37 "C with a solution containing 0.3 M sodium acetate and 10 mM Tris, pH 7.6, and the oligomers were desalted on a C-18 reversed-phase Sep Pak cartridge (27). The oligodeoxyribonucleosidemethylphosphonates were prepared on controlled pore glass supports in a Biosearch Model 8700 DNA synthesizer using standard methylphosphonamidite chemistry (27). The synthesizer was programmed to remove the 5'-terminal dimethoxytrityl group from the protected oligomer at the end of the syntheses, and the oligomer was deprotected and removed from the support by sequential treatment with a solution containing 853' 6 hydrazine hydrate, pyridine, and glacial acetic acid (1:24:6 v/v) for 16 h at room temperature, followed by treatment with a solution of 50% ethylenediamine in ethanol (v/v) for 6 h at room temperature (27). The oligomers were purified by DEAE cellulose chromatography and then by preparative C-18 reversed-phase chromatography on a 1.0 cm X 25 cm Ranin Microsorb 0 1993 American Chemical Society
Detection of Psoralen Cross-Link Sites
column using a gradient of 2% to 30 % acetonitrile in 0.05 Msodium phosphate, pH 5.8. The oligomers were desalted on a C-18 Sep Pak cartridge (27). The psoralen-derivatized oligodeoxyribonucleosidemethylphosphonates I1 and IV were prepared according to literature procedures (14, 28). The steps include (1) phosphorylating the oligomer using ATP and polynucleotide kinase, (2) converting the phosphorylated oligomer to its imidazolide derivative by treatment with l-ethyl3-[3-(dimethylamino)propyllcarbodiimide in 0.1 M imidazole buffer at pH 6.0, and (3)reaction of the imidazolide with 4'-[[N-(2-aminoethyl)aminolmethyll-4,5',8-trimethylpsoralen in 0.25 M lutidine hydrochloride buffer at pH 7.5. The psoralen-derivatized oligomers were purified by HPLC on a C-18 reversed-phase column using a 2 % to 30% gradient of acetonitrile in 0.05 M sodium phosphate buffered at pH 5.8. Preparation of the Photoadducts. A solution containing 0.1 pM 5'-[32Pl-end-labeledtarget DNA I or I11 and 5 pM psoralen-conjugated oligomer I1 or IV in 10 pL of 0.1 M sodium chloride, 50 mM Tris, pH 7.6, was irradiated in a borosilicate glass test tube for 15 min at 4 For each photoadduct, "C as previously described (7,14). a total of 16 reactions were run and then combined prior to purification. The photoadduct 1-11 or III-IV was purified by electrophoresis on a 14 cm X 16 cm, 20% polyacrylamide gel, which contained 7 M urea, run at 800 V for 2 h. The photoadduct was located by autoradiography and extracted from the gel as described above. Characterization of the Photoadducts. The target DNA and the photoadduct (1-2.5 X 104 dpm) were each treated in separate reactions under the following conditions: (1)50 pL of 1 M aqueous piperidine at 90 "C for 30 min; (2) 10pL of a solution containing 70 pM potassium permanganate, 0.1 M sodium chloride, 50 mM Tris, pH 7.6 (I and 1-11))or 70 pM potassium permanganate, 8 M urea (111and III-IV) for 30 min at 20 "C, after which the solution was treated with 2.5 p L of allyl alcohol and then treated with 40 pL of 1.25 M piperidine at 90 "C for 30 min; or (3) 10 pL of 88% formic acid for 5 min at 22 "C, after which the sample was precipitated with ethanol and the pellet treated with 50 pL of 1M piperidine at 90 "C for 30 min. When each reaction was completed, the piperidine was lyophilized, and the residue was lyophilized twice from 10 pL of water. The residue was dissolved in 6 pL of 90% formamide and the sample was electrophoresed on a 30 cm X 40 cm, 20 % acrylamide sequencing gel containing 7 M urea run at 1800 V for 2 h. The gel was autoradiographed at -80 "C using intensifying screens.
RESULTS AND DISCUSSION The sequences of the oligodeoxyribonucleotidetargets and their complementary oligodeoxyribonucleoside methylphosphonates which are conjugated with 4'- [ [N-(2aminoethy1)aminolmethy1]-4,5',84rimethylpsoralen,(ae)AMT, are shown in Figure 1. Target oligomer I contains a single thymidine a t position 16 which can serve as a site for reaction with the (ae)AMT group of 11. Irradiation of a solution of 5'-[32Pl-I and I1 gives a cross-linked adduct (1-11) whose gel electrophoretic mobility is less than that of I as is shown in lanes 4 and 5 of Figure 2A. This photoadduct most likely results from a 2 + 2 cycloaddition reaction between the pyranone ring of the (ae)AMTgroup and the 5,g-double bond of a T-16 in the target. Because of steric constraints, it appears highly unlikely that photocycloaddition occurs through the furan ring of (ae)AMT. We found that piperidine hydrolysis could be used to locate psoralen cross-linking sites. Previous experiments
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1 10 20 0-GCTCCAATTGACAMTM 3O TG-'
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Figure 1. Psoralen-conjugated oligonucleoside methylphosphonates and their oligo DNA targets. The underline indicates methylphosphonate linkages.
have shown that methylphosphonate linkages are readily hydrolyzed in aqueous piperidine solution, even at 37 "C (29). Thus, treatment of photoadduct 1-11 with piperidine at 90 "C (lane 7) completely cleaves the methylphosphonate portion of the photoadduct to give a "truncatedn photoadduct which contains only phosphodiester linkages and whose mobility is slightly less than that of strand I. In addition, a single 5'- [32Pl-labeledoligonucleotide is released whose mobility is consistent with cleavage at T-16, the psoralen cross-linksite. In these experiments, we found it important to use freshly prepared [32P]-end-labeled oligonucleotide in order to minimize nonspecific cleavage of the phosphodiester linkages. This and piperidine treatment conditions of 90 OC for 30 min resulted in a high signal to noise ratio, although the level of cleavage a t the psoralen cross-link site was relatively low. The photoadduct was also treated with potassium permanganate, which oxidizes the 5,6-double bonds of pyrimidines. When 1-11 was treated with potassium permanganate and then by piperidine (lane 8), cleavage occurred at T-16 and at C-2, T-3, and C-4, pyrimidines which are located outside or at the end of the methylphosphonate binding site. Cleavage was not observed at T-8 or T-9, residues which are base paired with the oligodeoxyribonucleosidemethylphosphonate and therefore are not subject to potassium permanganate oxidation (30). Comparison of lanes 1 and 8 shows that sequential treatment with potassium permanganate followed by piperidine provides a footprint of the oligomer binding site as well as the location of the cross-link site. Treatment of 1-11 with formic acid under conditions which cause depurination, followed by treatment with piperidine, resulted in cleavage at purine residues and cleavage at T-16. No cleavage was observed at T-16 when I is treated with formic acidlpiperidine although the same purine cleavage products were observed in both I and 1-11 at sites 5' to the cross-linksite (lanes 3 and 6). Cleavage of purines 3' to the cross-link site in 1-11 appeared to be reduced relative to that which occurred in I. The utility of this method was further demonstrated when applied to the characterization of the photoadducts formed by DNA target I11 and psoralen-conjugated oligodeoxyribonucleoside methylphosphonate IV. Although a single new slower moving product, III-IV, was observed after irradiation of I11 and IV, treatment of the photoadduct with piperidine resulted in cleavage at T-18 and T-19 in addition to the expected cleavage at T-17, as shown in lane 5 of Figure 2B. No cleavage of (2-7, T-11, C-12, (3-13, or T-14 was observed when III-IV was treated
Kean and Mlller
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Figure 2. Characterization of psoralen cross-link sites. Panel A: I + potassium permanganate/piperidine (lane 1);I +piperidine (lane 2); I + formic acid/piperidine (lane 3); I with no treatment (lane 4); 1-11 with no treatment (lane 5); 1-11 + formic acid/ piperidine (lane 6); 1-11 + piperidine (lane 7);and 1-11 + potassium permanganate/piperidine (lane 8). Panel B: I11 + potassium permanganate/piperidine (lane 1);I11 + piperidine (lane 2); I11 + formic acid/piperidine (lane 3); 111-IV + potassium permanganate/piperidine (lane 4); 111-IV + piperidine (lane 5); and IIIIV + formic acid/piperidine (lane 6). The reaction conditions and gel electrophoresis conditions are given in the Experimental Procedures section. The arrowheads indicate the psoralen crosslinking site(s) and the nucleotide sequences of the DNA targets are shown along the left side of each autoradiogram.
with potassium permanganate/piperidine,consistent with the expected binding site of the oligomer. This can be seen by comparing lanes 1and 4. Formic acid/piperidine treatment of the adduct gave the expected purine cleavage products in addition to cleavage at T-17, T-18, and T-19, as shown by comparing lanes 3 and 6. Cross-linking to T-17 is expected if the psoralen stacks on the last base pair formed between the oligomer/target and partially intercalates between T-16 and T-17 (14). A similar binding mode could occur between psoralen and T-18, if T-17 loops out, and between psoralen and T-19, if both T-17 and T-18 loop out. The apparent lack of cross-linking at T-16 suggests that photocycloaddition does not readily occur to the 3’-face of the T-16 nor does the psoralen intercalate between the last two base pairs of the duplex and react with the 5’-face of T-16. This behavior is in contrast to that seen in other systems, where cross-linking to the T of a terminal base pair was observed and probably reflects the effect of the sequence at the psoralen binding site on the position and extent of the cross-linking reaction (14).
Previous studies have shown that base treatment of psoralen cross-links results in cleavage of the cyclobutane ring and subsequent reversal of furan-side photoadducts, whereaspyranone-side cyclobutaneadducts do not appear to be cleaved by this treatment (17, 31). The strand scission at pyranone-side T photoadducts observed in our study may result from increased lability of the N-glycosyl bond caused by saturation of the 5,6-double bond of T. This increased lability would produce an abasic site which is subject to piperidine-catalyzed @elimination. The observation that identical cleavage products are formed by potassium permanganate/piperidine treatment of the target is consistent with this argument. The procedure described above provides a simple means to locate psoralen-thymidine photoadducts in DNA targets. Although we have used piperidine to characterize cross-links between psoralen-conjugated oligodeoxyribonucleoside methylphosphonates and single-stranded DNA targets, this method should also be applicable to detecting pyrone side adduct formed between other psoralenderivatized oligonucleotides or oligonucleotide analogs and DNA targets. ACKNOWLEDGMENT
This work was supported by a grant from the National Cancer Institute (CA 42762). We wish to thank Ms. Cynthia Cushman for assistance in preparing the oligomers and Dr. Bok L. Lee for helpful discussions. LITERATURE CITED (1) Gamper, H., Piette, J., and Hearst, J. E. (1984) Efficient formation of a crosslinkable HMT monoadduct a t the Kpn I recognition site. Photochem. Photobiol. 40, 29-30. (2) Houten, B. V., Gamper, H., Hearst, J. E., and Sancar, A. (1986) Construction of DNA substrates modified with psoralen a t a unique site and study of the action mechanism of ABC exonuclease on these uniformly modified substrates. J. Biol. Chem. 261,14135-14141. (3) Houten, B. V., Gamper, H., Holbrook, S. R., Hearst, J. E., and Sancar, A. (1986) Action mechanism of ABC excision nuclease on a DNA substrate containing a psoralen crosslink a t a defined position. Procd. Natl. Acad. Sci. (U.S.A) 83, 8077-8081. (4) Gamper, H. B., Cimino, G. D., and Hearst, J. E. (1987) Solution hybridization of crosslinkable DNA oligonucleotides to bacteriophage M13 DNA. Effect of secondary structure on hybridization kinetics and equilibria. J. Mol. Biol. 197,349382. (5) Shi, Y-B., and Hearst, J. E. (1987) Wavelength dependence for photoreactions of DNA-psoralen monoadducts. 1. Photoreversal of monoadducts. Biochemistry 26, 3786-3792. (6) Cheng, A., Houten, B. V., Gamper, H. B., Sancar, A., and Hearst, J. E. (1988) Use of psoralen-modified oligonucleotides to trap three-stranded Rec A-DNA complexes and repair of these cross-linked complexes by ABC exonuclease. J. Biol. Chem. 263, 15110-15117. (7) Lee, B. L., Murakami, A., Blake, K. R., Lin, S-B. and Miller, P. S. (1988) Interaction of psoralen-derivatized oligonucleoside methylphosphonates with single-stranded DNA. Biochemistry 27,3197-3203. (8) Lee, B. L., Blake, K. R., and Miller, P. S. (1988) Interaction of psoralen-derivatized oligodeoxyribonucleoside methylphosphonates with synthetic DNA containing a promoter for T7 RNA polymerase. Nucleic Acids Res. 16, 10681-10697. (9) Kean, J. M., Murakami, A., Blake, K. R., Cushman, C. D., and Miller, P. S. (1988) Photochemical cross-linking of psoralen-derivatized oligonucleoside methylphosphonates to rabbit globin messenger RNA. Biochemistry 27,9113-9121. (10) Pieles, U., and Englisch, U. (1989)Psoralen covalently linked to oligodeoxyribonucleotides: Synthesis, sequence specific recognition of DNA and photo-cross-linking to pyrimidine residues of DNA. Nucleic Acids Res. 17, 285-298.
Detection of Psoralen Cross-Link Sites
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