J . Am. Chem. SOC.1993, 115, 9303-9304
Efficient, Specific Interstrand Cross-Linking of Double-Stranded DNA by a Chlorambucil-Modified, Triplex-Forming Oligonucleotide Igor V. Kutyavin,' Howard B. Gamper, Alexander A. Gall, and Rich B. Meyer, Jr.
MicroProbe Corporation 1725 220th Street SE Bothell. Washington 98021
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9303 m
30
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1
111: IV: V: VI:
m
10
( I ) (a) Fedorova, 0. S.; Knorre, D. G.; Podust. L. M.; Zarytova, V. F. FEBS Lerr. 1988,228,273. (b) Vlassov, V. V.; Gaidamakov, S. A,; Zarytova. V. F.; Knorre, [I.G.; Levina, A. S.; Nikonova, A. A.; Podust, L. M.; Fedorova, 0.S. Gene 1988, 72.313. (c) Povsic, T. J.; Dervan, P. R. J . Am. Cbem. Soc. 1990,112,942X. (d) Shaw. J.-P.; Milligan, J. F.; Krawczyk,S. H.; Matteucci, M.J. Am. Chem. Soc. 1991. 113, 7765. (2) Young, S. L.; Krawczyk, S. H.; Matteucci, M. D.; Toole. J. J. h o c . Narl. Acad. Sci. U.S.A. 1991, 88, 10023. (3) (a) Takasugi, M.;Guendouz, A.;Chassignol, M.; Decout, J. L.; Lhomme. J.; Thuong, 3 . T.; Helene, C. Proc. Narl. Acad. Sci. U.S.A. 1991.88, 5602. (b) Duval-Valentin. G.; Thuong. N. T.; Helene, C. h o c . Natl. Acad. Sci. U.S.A. 1992, 89, 504. (c) Giovannangeli, C.; Thuong. N. T.; Helene, C. Nucleic Acids Res. 1992, 20, 4275. (d) Giovannangeli, C.; Rougee, M.; Garestier, T.; Thuong, N. T.; Helene, C . froc. Narl. Acad. Sci. U.S.A. 1992, 89,863 1. (e) Grigoriev, M.; Praseuth, D.; Guieysse, A. L.; Robin, P., Thuong. N. T.; Helene. C.; Harel-Bellan, A. Proc. Narl. Acad. Sci. U.S.A. 1993, 90, 3501. (4) Meyer, R. B.; Tabone. J. C.; Hurst, G. D.; Smith, T.M.; Gamper, H. J. Am. Cbem.Soc. 1989, I l l , 8517. ( 5 ) For instance, the reaction of a bromoacetamidopropylderivatizedODN with 1 m V cysteine is 90% complete after 1 h at 37 "C. (6) (a) Knorre. D. G.; Vlassov, V. V. Generico 1991.85, 53. (b) Knorre, D. G.; 7arytova. V. F. In Prospects for Anfisense Nucleic Acid Therapy of Cancer and AIDS; Wickstrom, E., Ed.; Wiley-Liss: New York, 1991; p 195.
40
r)
X IY = T; X = T; Y = Tp-O(CH,),NH-CIAmb X = CIAmb-HN(CH,),O-pT; Y = T X ICIAmb-HN(CH,),O-pT; Y = Tp-O(CH,),NH-CIAmb
ClAmb = p[(CICH,CH,),N]C,H,(CH,),CO-
Figure 1. ODNs used in this study. The double-stranded target formed by ODNs I and I1 contains a homopyrimidine/homopurinerun which differs by only two base pairs from the target used by Maher I1 et aI.l5
Reactive O D N
+
1
3'-TTTATGACCCTCTTTCCTClClTTTCCCCTGGGTTGCATA-S' S * - X C'T T T C'C'T C O T C'T T T T C'CC'CY 3 ' Ill-VI II S'-AAATACTGGGAGAAAGGAGAGAAAAGGGGACCCAACGTAT-3'
Received June 14, I993 Selective and irreversiblegene inactivation should be attainable by sequence-targeted interstrand cross-linkage of the genomic DNA. Oligodeoxynucleotides (ODNs) capable of forming sequence-specifictriple helices with double-stranded DNA targets and which bear alkylating groups for covalent modification of the target strands offer a means of testing this hypothesis. Covalent modification of one strand of the targeted duplex has been demonstrated for polypyrimidine ODNs conjugated to a single reactive group.' These ODNs irreversibly react with the homopurine-containing strand within the triplex to block transcription* and possibly replication. ODNs which react with only one strand of the duplex target would be expected to exert transitory effects due to DNA repair. Triple-helix-forming ODNs which covalently react with both strands of a DNA target might mutationally inactivate the targeted gene through an error-prone repair pathway, thus permanently inhibiting gene expression. ODNs bearing the pendant bifunctional photoactive cross-linker psoralen can meet this specification3 but will be limited to topical applications in therapeutics due to the requirement for activation by ultraviolet light. We and others have found that electrophilic moieties like cy-haloacetamide~'~,~ react too readily with biological amines and thiols.' Nitrogen mustards offer the advantage of low reactivity until the formation of a highly reactive aziridinium ion and have been used in ODN-directed duplex6and monofunctional cross-linking. We report here that triple-helix-forming ODNs modified at the 3'- and 5'-ends with the clinically used nitrogen mustard chlorambucil are able to rapidly alkylate both strands of a DNA target with excellent efficiency and specificity. The ODNs used in this study are shown in Figure 1. The double-stranded target formed by I I1 is a 40-mer, with I1 containing a 23-base-long A-rich homopurine tract. Polypyrimidine 20-men 111-VI form the triplex by binding, with Hoogsteen H-bonding, to the purines of I1 within the I-II duplex. Chlorambucil (ClAmb) was coupled to IV-VI through 3' and/or
10
1
IV
v
VI
IV
v
VI
I
Figure 2. Analysis of reaction mixtures containing 5'-32P-labeled duplex I + I1 and reactive ODNs IV-VI. Preformed duplex (2 X 10-* M labeled strand and 4 X IO-* M unlabeled strand) was incubated for 6 h at 37 O C with triple-helix-forming ODNs IV-VI (1 X lO-' M) in 140 mM KCI, 10 mM MgC12.1 mM spermine, and 20 mM HEPES, pH 6.0. Aliquots were analyzed on an 8% denaturing polyacrylamide gel. The first lane in each series is a control reaction mixture which lacked any triplexforming ODN. The structures of the products are schematically represented to the left of the gel.
5'-aminohexyl phosphate groups' added to theend(s) of the ODN at the time of synthesis.* All of the triple-helix-forming ODNs contained 5-methylcytosine (C+) residues to stabilize triplex format i ~ n . ~ Alkylation of the targeted duplex strands by reactive ODNs IV-VI was monitored by denaturing polyacrylamide gel electrophoresis (Figure 2). Two classes of reaction products were resolved, a mono-cross-linked product linking the reactive ODN with one target strand, and a bis-cross-linked product linking it with both target strands. These results show that, for this target, (7) Thereagent for the 5'-aminohexyl modification iscommerciallyavailable from Glen Research, Sterling, VA. The method for the 3'-aminohexyl modifications is as described: Petrie, C. R.; Reed, M. W.; Adams, A. D.; Meyer, R. B., Jr. Bioconjugate Cbem. 1992, 3, 85-87. (8) The coupling reaction employed the 2.3.5.6-tetrafluorophenyl ester of chlorambucil. Modified ODNs were purified by reverse-phase chromatography, concentrated by extraction with 1-butanol, and precipitated in acetone containing 2% LiC104. A11 manipulations were performed in ice to minimize reaction of the bis-N,N-(2-chloroethyl)amineresidue. The modified ODNs were stored in aqueous solution at -70 "C. AI! of the chlorambucil-modified oligomers showed a single band on reverse-phase HPLC. Reaction of these modified ODNs with 1 .M tetraethylenepentamine in water for 6 h at 37 "C, followed by electrophoreticanalysis.indicated that >85%oftheODN possessed the expected alkylating ability. (9) (a) Povsic, T. J.; Dervan. P. €3. J. Am. Chem. Soc. 1989, I l l , 3059. (b) Collier, D. A.; Thuong, N. T.; Helene, C. J. Am. Cbem. Soc. 1991,113, 1457.
OOO2-7863/93/1515-9303$04.00/00 1993 American Chemical Society
Communications to the Editor
9304 J. Am. Chem. Soc., Vo!. 115, No. 20. 1993 32P-ODN
7I1 7
Reactive ODN
I\’
A
+ G G A A T A C T
-G -G -G A G
A A A G G
A G A G
A A A A
*G -G +G *G A C C C A A C G T A 3’7
v
I ,-i IV VI
VI
A + T
G
T 3’ T T A T G A C C C ’X T C+ C T T T 1 T T C+ C C+ C T T C+ C T T C+ C T T T T T T T T C+ C C+C C+C C+C T
Ge GGT T G C A T 5‘
Fipre3. Determination of the sitesof alkylation in 5’-32P-labeled duplex strands I and I1 by reactiveODNs IV-VI. The majorcross-linked products generated by each reactive ODN were isolated by denaturing PAGE (see Figure 2), and the products were eluted from the appropriate sections of the gel which had been cut out. They were treated for 15 min at 95 OC in 1 M pyrrolidine16 tocleave the DNA backboneat thesitesofalkylation, evaporated twice in water, and run on an 8% denaturing polyacrylamide gel. The positions of alkylation are indicated in the triple strand to the left of the gel. For reactive ODN VI, only the interstrand bis-crosslinked products were analyzed; hence, no cleavage at G26-G29 in target strand I1 is observed with this ODN. Because of the low level (
