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J. Am. Chem. SOC. 1995, 117, 3629-3630
Sequence-Specific Photocleavage of DNA by an Expanded Porphyrin with Irradiation above 700 nm
LuTx
1
2
sapphyrin
3
4
5
6
3629
TMPyPH2 7
8
9
Dmen Magda,*J Meredith Wright,' Richard A. Miller,' Jonathan L. Sessler,*.t and Petra 1. Sansom' Phannacyclics, Incorporated, 995 Easr Arques Sunnyvale, California 94086 Deparimeni of Chemistry and Biochemisrry Universiry of Texas ai Ausiin, Ausiin, Texas 78712 Received December 23, 1994
The development of chemically modified oligonucleotides as a means of regulating gene expression is a topic of current widespread in1erest.l One approach being pursued involves using antisense agents containing photoactivatable groups.2-6 Such a strategem offers the advantage of providing sequencespecific reagents that are inert in the dark, yet subject to being "switched on" via irradiation at a particular wavelength. Currently, oligonucleotide conjugates containing azido derivatives? ellipticines? psoralens: proflavins? and porphyrin moieties6 are known. All of these derivatives modify DNA when irradiated at wavelengths below 700 nm. For use in vivo, however, pbotoactivation in the 700-900 nm region would be preferable as it is here that bodily tissues are most transparent? In this communication, we describe the photocleavage capabilities of two expanded porphyrins, the lutetium(II1) texaphyrin complex (LuTx) 1 and the nonmetalated sapphyrin 4, both of which cleave DNA upon irradiation at wavelengths above 700 nm. The more efficacious of these two chromophores, LuTx 1, has also been incorporated into oligonucleotide conjugates (e.&, 3). The latter provide the fin1example of oligonucleotidedirected photocleavage of DNA with irradiation above 700 nm.
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Figure 1. Photograph of a 0.8% agarose gel containing ethidium bromide showing the results of a pBR 322 plasmid DNA cleavage sNdy. In accord with the general procedure of refs 8 and IO. the DNA was irradiated in quartz cuvettes in the presence of either LuTx 1, sapphyrin 4, or TMPyPHz. Irradiation was effected at loom temperature using light from a high-pressure xenon lamp (Oriel) passed through a 700 nm filter (CVI Laser). The sample received approximately 280 mW1cm'. The following conditions pertained. DNA 20.4 ngIpL (all cases). Lanes I. 4, 7: 0 p M chromophore, r = 60 min. Lanes 2, 5. 8: 4 @M chromophore, r = 0 min. Lanes 3.6, 9 4 pM chromophore, r = 60 min.
meso-tetrakis(4-N-methylpyridyl)porphine(TMF'YPH~),~.~ using a general pBR 322 plasmid DNA DNA cleavage was followed by monitoring of the conversion of supercoiled (form I) plasmid DNA to the nicked circular (form 11) DNA. At wavelengths above 300 nm, all three compounds showed efficient photocleavage (cf. supplementary material). When, however. the shorter wavelengths were blocked out with a 700 nm filter, cleavage efficiencies of 870, 17%, and 93% were recorded for the porphyrin control, sapphyrin 4, and LuTx 1. respectively (Figure I)."." In order to test the ability of LuTx to cleave DNA within a double-stranded helix, the LuTx complex 2 was conjugated with two 2'-O-methyl RNA 15-mers (Figure 2. 3A and 3B).I3-l5 Synthetic DNA 36-mers 5 and 6 were selected as substrate^,'^ each complementary in sequence to only one of the LuTx
(4) (a) Bhan, P.; Miller, P. S.Biocmjugare Chem. 1990. 1. 82-88. (b) Team I.; Wollenzien. P. Nucleic Acids Res. 1989, 17. 3359-3312. (c) Pieles. U.; Englixh, U. Nucleic Acids Res. 1989. 17.285-299. (d) Takasugi, M.; Guendour. A,: Chassignol. M.; Decout. J. L.: Lhomme, I.; Thuong. N. T.: Hdilne. C. Proc. Nafi. Acad. Sci. U.S.A. 1991. 88, 5602-5606. ( 5 ) Praseuth, D.; Le Doan, T.: Chassignol, M.: Decout. 1.-L.; Habhoub, N.; Lhomme. I.: Thuong, N. T.: Hellne. C. Biochmi.sfq 1988.27, 303 I 3038. (6) (a) Mastmzro. L.; Woisard, A,: Ma, D. D. F.; Rirzarelli. E.; Favre, A,: Le Doan. T. Phofochem. Phorobiol. 1994.60,316-322. (b) Fedorova, 0. S.; Savitskii. A. P.; Shoikhet. K. G.: Ponamarev. G. V. FEES Lrrr. 1990. 259. 335-337. (c) Vlassov, V. V.; Deeva, E. A,; Ivanova. E. M.; Knorre. D. G.: Maltseva, T. V.: Frolova, E. 1. Nucleosides Nudeorides 1991, IO. OH 64-643. (d) Le Doan, T.: haseuth. D.: Permuault. L.; Chassignol, M.: Thuong. N. T.; Hillne, C. Bioconjugore Chem. 1990, 1. 108-1 13. 1 R p = R3 = OCH~CHZCH~OH 4 R = CH~CON(CHZCH~OH)~ (7) Wan. S.: Parrish, J. A,: Anderson, R. R.; Madden. M. Phmchem. Phofobioi. 1981. 34. 679-681. 2 R2 = OCHzCOzH, RJ = H (8) Praseuth, D.; Gaudemer. A,: Verlhac, J.-B.; Kraljic, 1.: Sisseff. 1.; 3 R2 = OCH2CO-RNA (Z-OMe). Guilld. E. Phofochem. Phorobiol. 1986.44. 117-724. RQ-H (9) meso-Tetrakir(4-N-methylpyridyl)porphine was purchased from Aldrich Chemical Company. (10)Croke. D. T.: Permuault. L.; Sari, M. A,: Battioni. 1.-P.: Mansuy. LuTx 1 and sapphyrin 4 were screened for their photoD.: Hellne. C.: Le Doan. T. J. Phofochem. Phorobiol., B 1993. 18.41-50. cleavage capabilities against the known photocleaving agent ( I I ) The factors influencing the relative photocleavage efficiencies of the different chromophores are complex and could reflect such effects as Phannacyclics. Inc. differences in absorption maxima. singlet oxygen quantum yields. binding University of Texas at Austin affinities, etc. (I)(a) Uhimann, E.; Peyman. A. Chem. Rev. 1990. 90,543-584. (b) (I2)Cleavageyields were measured by densitometry and calculated as Hellne, C.: Taulme. 1.-J. Biochim. Biophys. Acto 1990,1049.99-125. (c) in ref 8. Godchild. I. Bioconjugofe Chem. 1990. I, 165-187. (d) Englisch, U.: (13) 2'-OMe derivatives were chosen because they were thought to be Gauss. D. H. Anaew. Chem.. Inf. End. Ed. 1991.30.613-629. le) Pm.w"s more useful for in vivo work. Unmodified DNA conjugates were also made. ~~r~~~~ for Anfisense N;cleic Acid Therafi of Cancer and AiDX Wicksuom, E., However. the conjugates prepared using 2'-O-methyl RNA proved more Ed.; Wiley-Liss. Inc.: New York, 1991. (0 Anfisense Research and effective. Cf. footnote 22. Appiicafions; Crooke, S. T.. Lebleu. B.. Eds.; CRC press: Boca Raton, (14) 2'-OMe derivatired oligoribonucleotide amines modified on the 5' FL. 1993. end were purchased from Keystone Laboratories. Inc., as were the DNA (2) (a) Le Doan. T. ;Permuault. L.: Raseuth. D.: Habhoub. N.: kcout. 36-mer substrates. All conjugates and substrates were PAGE purified and I.-L , nuong. N. 7.. Lhomme. I.: HtlCnc. c NUCIW A ~ GRPV. J ~ i987.15: ethanol precipitated prior to use. 774Y-7760 (hj Levma. A. S.: Benmsku. M. V.; Vcnpmmobs. A. G . : (15)Magda. D.: Miller. R. A.; Sessler.1.L.: Iverson, B. L. 1. Am. Chem. Tnn~ 116 Dobrikor. M I : Repkuva. M. N , 7agtova. V F. B # o d ~ # m #1993. e 75. " I.1._ ", 7A70-7ddn 7 c 3 , &< &,. 116) Photochemical reaction, wcrc c m c d out b) trcmng 20pL sampler (3) Permuault. L.; Asseline. U.: Rivalle. C.; Thuong, N. T.; Bisagni. E.: thmuph the
