Bioconjugate Chem. 1996, 7, 541−544
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Antisense Sequence-Directed Cross-Linking of DNA Oligonucleotides by Mitomycin C Helena Maruenda† and Maria Tomasz* Department of Chemistry, Hunter College, The City University of New York, New York, New York 10021. Received May 31, 1996X
Oligodeoxyribonucleotides (ODNs) conjugated with mitomycin C (MC) via (-CH2-)n tethers of different lengths (n ) 6, 12) to their terminal 5′-phosphate were synthesized, and their interaction with target complementary single-stranded DNA oligonucleotides was investigated. MC, a clinically used natural anticancer drug, is known to act as a bioreductive alkylating agent of duplex DNA with a remarkable preference for 5′-d(CG) sequences. The usual enzymatic bioreductive techniques known to trigger MC to alkylate DNA were employed in the reaction between the MC-oligonucleotide conjugates and their targets radiolabeled by 32P at their 5′-phosphate. A slow-moving radiolabeled product, detected by polyacrylamide gel electrophoresis using phosphorimaging techniques, was obtained in 15-25% yield with complementary DNA as target. Formation of these products was dependent upon complementary duplex formation. Evidence is presented that the DNA target is alkylated by the mitomycin C moiety of the ODN conjugate at the 2-amino group of a guanine base. These findings suggest that the MC-ODN conjugates may be useful specific inhibitors of cellular or viral gene expression. To our knowledge this is the first report on ODN conjugates of a reductively activated drug of known therapeutic value.
The attachment of DNA-reactive agents to oligodeoxyribonucleotides (ODNs)1 has led to conjugate molecules capable of inhibiting biosynthesis of specific proteins (1, 2) and blocking of expression of specific genes (3, 4). Examples of such agents are the photoactivatable compounds psoralen (5) and p-azidoacetophenone (6), chemically reactive DNA cross-linkers of the N-mustard class (7), R-halocarbonyl compounds (8), and photoactivatable and reductively activatable naphthoquinone derivatives (9). The specificity of biochemical action of a reactive agent-ODN conjugate is based on targeting the reaction to a specific nucleic acid sequence by base complementarity between the target and the agent-ODN conjugate. One of the cross-linking agents not yet exploited in this area is the antitumor antibiotic and clinically used cancer chemotherapeutic drug mitomycin C (MC,1 1a). We report the synthesis of covalent MC-ODN conjugates as potential sequence-specific nucleic acid-inactivating agents. It is shown that MC conjugated to ODNs at their 5′-end specifically alkylates complementary DNA targets. Reduction of MC activates the C1-aziridine and C10carbamate positions to alkylate DNA (10). Both monofunctional (2a and 3a) and bifunctional alkylation products of DNA (4a) are formed. The single target of alkylation by MC is the 2-amino group of guanine residues in the DNA minor groove. A conspicuous preference is displayed by MC for alkylation and crosslinking of guanines in the CpG sequence (10). A versatile way of derivatizing the C7-amino substituent of MC is through its analog, mitomycin A (1b) (11). † Present address: UC Berkeley, Lawrence Berkeley Laboratories, Berkeley, CA 94720. X Abstract published in Advance ACS Abstracts, September 1, 1996. 1 Abbreviations: ODN, oligodeoxyribonucleotide; DPAGE, denaturing polyacrylamide gel electrophoresis; MC, mitomycin C; equiv, equivalent; dG, 2′-deoxyguanosine; dR, 2′-deoxyribos1′-yl; TBE, Tris-borate-EDTA.
S1043-1802(96)00054-7 CCC: $12.00
Chart 1
Hence, simple condensation of the latter, with oligonucleotides containing a primary aliphatic amino group tethered to their 5′-phosphate group (5)2 yielded MC-ODN conjugates 6-8 (Scheme 1). The products were purified by reversed-phase HPLC; their UV spectra are exemplified in Figure 1. Rigorous proof for the structure of 7 was provided by degradation with snake venom diesterase and alkaline phosphatase to yield 1c, which was identified by comparison with an authentic synthetic sample (11). The 15-mer MC-ODN conjugates 6-8 used in this study are listed in Chart 2. Molecular modeling of 6 and its shorter (-CH2-)n tether length variants annealed to the complementary 18-mer target ODN 9a suggested that n ) 10 gave optimal linker length for cross-linking of conjugates to the targeted guanine G-15.3 We opted for the commercially available amino linker n ) 12 instead, to synthesize 6.2 In addition, we also synthesized two MC-ODN conjugates 7 and 8 with 2 “5′-amino linker” oligodeoxyribonucleotides (5) were synthesized by a DNA synthesizer (ABI Model 380B) using cyanoethyl phosphoramidites (ABI) and “5′-amino linkers” (Glen Research, Sterling, VA) and were purified by reversed-phase HPLC. 3 Personal communication with Dr. David Langley, BristolMyers Squibb Co., Wallingford, CT.
© 1996 American Chemical Society
542 Bioconjugate Chem., Vol. 7, No. 5, 1996
Figure 1. UV spectrum of the mitomycin-ODN conjugate 6 and various mitomycin-deoxyguanosine adducts, determined by diode array scanning of eluates from HPLC: (a) conjugate 6; (b) deoxyguanosine, 2,7-diaminomitosene, M2; (c) adduct 3a (1′′-R); (d) adduct 3c (I). Scheme 1. Synthesis of MC-ODN Conjugates 6-8
Maruenda and Tomasz
Figure 2. Cross-linking of MC-ODN conjugates to ODN targets, detected by DPAGE. Complete reaction mixture: MCODN conjugates (22 µM) were annealed with 5′-32P-labeled target ODNs (2 equiv) in 100 mM Tris buffer, pH 7.5 (25 µL), then treated under anaerobic conditions with NADPH (2 mM) and NADPH-cytochrome c reductase (30 units/mL) in a total volume of 50 µL, and incubated at 37 °C for 1 h. After phenol/ CHCl3 extraction of the reaction mixture, the oligonucleotide material was precipitated, centrifuged, and redissolved in 7 M urea-containing loading buffer according to standard protocols (12), followed by electrophoresis in 20% polyacrylamide-7 M urea gel, using 1 × TBE as buffer. (a) Lanes 1-3 (n ) 12 system): lane 1, complete reaction mixture of 6 with 9a minus enzyme; lane 2, complete reaction mixture of 6 with 9a; lane 3, complete reaction mixture of 6 and noncomplementary target ODN d(5′-TCACGACTGAGATCGGAG-3′). Lanes 4-6 (n ) 6 system) (a): lane 4, complete reaction mixture of 7 and 10a minus enzyme; lane 5, complete reaction mixture of 7 and 10a; lane 6, complete reaction mixture of 7 and noncomplementary target ODN d(5′-TCACGACTCCGTATCGCG-3′). Lanes 7 and 8 (n ) 6 system) (b): lane 7, complete reaction mixture of 8 and 11 minus enzyme; lane 8, complete reaction mixture of 8 and 11. Chart 2. Stucture of MC-ODN Conjugates 6-8 (Top Strands) and Their Complementary Target ODNs (Bottom Strands)a
shorter (n ) 6) linkers, regardless of the results of the modeling, and examined the cross-linking abilities of all three conjugates as follows. Conjugates 6-8 were annealed with 2 equiv of their respective complementary target strands 9a, 10a, and 11 radiolabeled by [32P]phosphate at their 5′-end (12). The MC was then reductively activated with NADPH-cytochrome c reductase/NADPH under anaerobic conditions (13). The resulting alkylation of the target strand was monitored by denaturing polyacrylamide gel electrophoresis (DPAGE) using phosphorimaging for detection of radiolabeled material. In addition to unreacted target DNA, the formation of a slower migrating species was observed in 25% yield for the n ) 12 system and in 15% yield for both n ) 6 systems (Figure 2). The mobility of this band paralleled the mobility of a marker 15-mer/ 18-mer duplex cross-linked by free MC (12) according to our standard procedure (14) (data not shown). Noncomplementary target sequences provided no detectable cross-linked products with 6 and 7 (lanes 3 and 6), confirming that the cross-linking is specific to complementary duplex structures, although the effect of mismatched basepairs on the specificity was not tested. Efficiency of Various Reductive Activation Systems. Chemical reduction (Na2S2O4, NaBH4) (14) was ineffective to induce cross-linking of the conjugates to their targets, while NADH-cytochrome c reductase and xanthine oxidase (13) were as efficient as NADPHcytochrome c reductase. Gluthathione reductase was inactive (data not shown).
a All target ODNs were radiolabeled by [32P]phosphate at their 5′-ends by standard procedures (12).
Characterization of Mitomycin-DNA Adducts. Unlabeled cross-linked products were isolated from electrophoresis gels4 and were digested with snake venom diesterase and alkaline phosphatase. HPLC of the digests yielded several different MC adducts marked
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Bioconjugate Chem., Vol. 7, No. 5, 1996 543
Site Specificity of Cross-Linking. The cross-linking is dependent on sequence complementarity of the conjugate and its target. No absolute selectivity exists for any of several Gs within the “reach” of the tethered mitomycin, however, as seen by base substitution experiments: Cross-linking yields of 6 + 9a, 6 + 9b, and 6 + 9c systems were 26%, 26%, and 18%, respectively. This indicates that G15 is definitely an alkylation site but that G16 and/or G18 are also alkylated when G15 is replaced by I15. Similarly, 7 + 10a yielded 15% cross-link, while 7 + 10b yielded 8%, implicating G14 and/or G18 as additional reaction sites, besides G16. The results herein demonstrate that the alkylating activity of MC can be targeted to specific DNA via antisense recognition between the target sequence and the MC-oligonucleotide conjugate. The requisite bioreductive activation of MC is accomplished in its ODNconjugated form by the same reductase systems known to activate free MC in vitro and in vivo (10). ACKNOWLEDGMENT Figure 3. HPLC of enzymatic digests of the reaction products between mitomycin-ODN conjugates and their complementary target ODNs: (a) digest of the cross-linked fraction from the reaction of 6 with 9a; (b) digest of the cross-linked fraction from the reaction of 7 with 10a; (c) digest of the non-cross-linked fraction of the same. HPLC: Rainin ODS (C18) column (0.45 × 15 cm), eluted with 9-54% CH3CN in 0.03 M NH4OAc in 90 min for (a) and with 6-36% CH3CN in 0.03 M NH4OAc in 90 min for (b) and (c); flow rate ) 1 mL/min.
I-IV in parts a and b of Figure 3. Digestion of the isolated faster moving, non-cross-linked electrophoretic bands also yielded adducts, most likely derived from selfstrand alkylation of 6 and 7. These are marked II′- IV′ in Figure 3c. Two of them (III′ and IV′) were identical with III and IV, respectively. All adducts were characterized as adducts of a 7′′-(alkyl)aminomitosene and deoxyguanosine, on the basis of their UV spectra, which display (e.g. Figure 1d) a clear combination of these two characteristic chromophores (Figure 1c). The UV spectrum of authentic 3a (1′′-R; Figure 1b) is essentially identical with the spectra of all of our adducts (see e.g. Figure 1d).5 Furthermore, the UV spectra uniquely specify N2- or N1-substituted dG (15). The latter is discounted, however, since G-N1 substitution, in contrast to G-N2 substitution, has never been observed by mitomycins (10). ESIMS(+)6 confirmed the structure of I as monoadduct 3c, II and II′ as monoadducts 3b (1′′stereoisomers), and III and III′ (identical) as monoadduct 2b. IV (identical with IV′) yielded no mass data. It must be a monoadduct since it is isolated from both the crosslinked and non-cross-linked fractions. It may be the 1′′stereoisomeric monoadduct 2b. In summary, it is shown that the cross-linking takes place through monofunctional alkylation of the N2 of guanine by the conjugated MC, the same as by free MC (10). 4 The cross-linked DNA was isolated preparatively by denaturing gel electrophoresis (20%, 7 M urea, 1 × TBE buffer) and located by UV shadowing technique. This gel slice was crushed and soaked in deionized water for 12 h. The slurry was filtered, and the solution was chromatographed over a Sephadex G-25 column, in 0.02 M NH4HCO3. 5 Except for a 6-8 nm red shift of the 314 nm band, due to the change from the 7′′-amino to the 7′′-alkylamino substituent. 6 ESIMS results (m/z): 3c (I), 755 [(MH)+], 678 [(MH - dR)+], 253 [(MH3)3+]; 3b (II), 673 [(MH)+ for hydroquinone]; 3b (II′), 673 [(MH)+ for hydroquinone]; 2b (III, III′), 627 [(MH)+].
These studies were supported by NIH Grant CA28681 and a “Research Centers for Minority Institutions” award (RR03037) from the Division of Research Resources. We thank Dr. David Langley, Bristol-Myers Squibb Co., Wallingford, CT, for fruitful discusssions. The mass spectroscopy was performed by Robert Rieger from the MS Facility, Chemistry Department, SUNY at Stony Brook. LITERATURE CITED (1) Pascolo, E., Hudrisier, D., Sproat, B., Thuong, N. T., and Toulme´, J. J. (1994) Relative contribution of photo-addition, helper oligonucleotide and RNase H to the antisense effect of psoralen-oligonucleotide conjugates, on in vitro translation of Leishmania mRNAs. Biochim. Biophys. 1219, 98-106. (2) Levis, J. T., and Miller, P. S. (1994) Properties of exonuclease-resistant, psoralen-conjugated oligodeoxyribonucleotides in vitro and in cell culture. Antisense Res. Dev. 4, 231241. (3) Grigoriev, M., Praseuth, D., Guieysse, A. L., Robin, P., Thuong, N. T., He´le`ne, C., and Harel-Bellan, A. (1993) Inhibition of gene expression by triple helix-directed DNA crosslinking of specific sites. Proc. Natl. Acad. Sci. U.S.A. 90, 3501-3505. (4) Degols, G., Clarenc, J. P., Lebleu, B., and Le´onetti, J. P. (1994) Reversible inhibition of gene expression by a psoralen functionalized triple helix forming oligonucleotide in intact cells. J. Biol. Chem. 269, 16933-16937. (5) Kean, J. M., and Miller, P. S. (1993) Detection of psoralen cross-link sites in DNA modified by psoralen-conjugated oligodeoxyribonucleotide methylphosphonates. Bioconjugate Chem. 4, 184-187. (6) Praseuth, D., Perrouault, L., Le Doan, T., Chassignol, M., Thuong, N., and Helene, C. (1988) Sequence-specific binding and photocrosslinking of alpha and beta oligodeoxynucleotides to the major groove of DNA via triple-helix formation. Proc. Natl. Acad. Sci. U.S.A. 85, 1349-1353. (7) Vlassov, V. V., Zarytova, V. F., Kutyavin, I. V., Mamaev, S. V., and Podyminogin, M. A. (1986) Complementary addressed modification and cleavage of a single stranded DNA fragment with alkylating oligonucleotide derivatives. Nucleic Acids Res. 14, 4065-4076. (8) Meyer, R. B., Tabone, J. C., Hurst, G. D., Smith, T. M., and Gamper, H. J. (1989) Efficient specific cross-linking and cleavage of DNA by stable synthetic complementary oligodeoxyribonucleotides. J. Am. Chem. Soc. 111, 8517-8519. (9) Chatterjee, M., and Rokita, S. E. (1994) The role of a quinone methide in the sequence specific alkylation of DNA. J. Am. Chem. Soc. 116, 1690-1697.
544 Bioconjugate Chem., Vol. 7, No. 5, 1996 (10) Tomasz, M. (1994) The mitomycins: Natural cross-linkers of DNA. Molecular Aspects of Anticancer Drug-DNA Interactions (S. Neidle and M. Waring, Eds.) Vol. 2, pp 312-349, CRC Press, Boca Raton, FL. (11) Iyengar, B. S., Lyn, H.-Y., Cheng, L., and Remers, W. A. (1981) Development of new mitomycin C and porfiromycin analogues. J. Med. Chem. 24, 975-981. (12) Maniatis, T., Fritsch, E. F., and Sambrook, J. (1989) Molecular Cloning. A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. (13) Tomasz, M., Chowdary, D., Lipman, R., Shimotakahara, S., Veiro, D., Walker, V., and Verdine, G. L. (1986) Reaction
Maruenda and Tomasz of DNA with chemically or enzymatically activated mitomycin C: Isolation and structure of the major covalent adduct. Proc. Natl. Acad. Sci. U.S.A. 83, 6702-6706. (14) Borowy-Borowski, H., Lipman, R., Chowdary, D., and Tomasz, M. (1990) Duplex oligodeoxyribonucleotides crosslinked by mitomycin C at a single site: Synthesis, properties, and cross-link reversibility. Biochemistry 29, 2992-2999. (15) Singer B., and Grunberger, D. (1983) Molecular Biology of Mutagens and Carcinogens, pp 295-334, Plenum Press, New York.
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