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. 1997 Jun 15;25(12):2352–2358. doi: 10.1093/nar/25.12.2352

Synthesis and evaluation of oligodeoxyribonucleotides containing an aryl(trifluoromethyl)diazirine moiety as the cross-linking probe: photoaffinity labeling of mammalian DNA polymerase beta.

T Yamaguchi 1, K Suyama 1, K Narita 1, S Kohgo 1, A Tomikawa 1, M Saneyoshi 1
PMCID: PMC146739  PMID: 9171085

Abstract

Photolabile 2'-deoxy- E -5-[4-(3-trifluoromethyl-3 H-diazirin-3-yl)styryl]uridine and its protected phosphoramidite derivatives have been synthesized and introduced into DNA oligomers through solid-phase DNA synthesis. The (trifluoromethyldiazirinyl)stylyl moiety of this nucleoside was found to be sufficiently stable for automated DNA synthesis. In addition, this moiety was found to be stable at 60 degrees C in aqueous solution under the annealing conditions for duplex formation with complementary strands, since >95% of the photolabile nucleoside remained after heating for 1 h. The oligo(dT) 15mer analog bearing the photolabile residue was activated/decomposed by near-UV irradiation. In photoaffinity cross-linking experiments with recombinant rat DNA polymerasebeta, constituted from a 40 kDa polypeptide, using oligo(dT) 15mer analogs bearing the photolabile residue near the 3'-terminus, a covalently bound complex of 45 kDa was obtained in the presence of complementary templates. Thus it was demonstrated that our method for synthesis of photolabile oligodeoxyribonucleotides may be useful for studies of DNA-related enzymes and DNA binding proteins.

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Selected References

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  1. Bartholomew B., Tinker R. L., Kassavetis G. A., Geiduschek E. P. Photochemical cross-linking assay for DNA tracking by replication proteins. Methods Enzymol. 1995;262:476–494. doi: 10.1016/0076-6879(95)62039-7. [DOI] [PubMed] [Google Scholar]
  2. Brunner J. New photolabeling and crosslinking methods. Annu Rev Biochem. 1993;62:483–514. doi: 10.1146/annurev.bi.62.070193.002411. [DOI] [PubMed] [Google Scholar]
  3. Brunner J., Senn H., Richards F. M. 3-Trifluoromethyl-3-phenyldiazirine. A new carbene generating group for photolabeling reagents. J Biol Chem. 1980 Apr 25;255(8):3313–3318. [PubMed] [Google Scholar]
  4. Capson T. L., Benkovic S. J., Nossal N. G. Photochemical cross-linking of DNA replication proteins at primer terminus. Methods Enzymol. 1995;262:449–456. doi: 10.1016/0076-6879(95)62036-2. [DOI] [PubMed] [Google Scholar]
  5. Capson T. L., Benkovic S. J., Nossal N. G. Protein-DNA cross-linking demonstrates stepwise ATP-dependent assembly of T4 DNA polymerase and its accessory proteins on the primer-template. Cell. 1991 Apr 19;65(2):249–258. doi: 10.1016/0092-8674(91)90159-v. [DOI] [PubMed] [Google Scholar]
  6. Czarnecki J., Geahlen R., Haley B. Synthesis and use of azido photoaffinity analogs of adenine and guanine nucleotides. Methods Enzymol. 1979;56:642–653. doi: 10.1016/0076-6879(79)56061-3. [DOI] [PubMed] [Google Scholar]
  7. Date T., Yamaguchi M., Hirose F., Nishimoto Y., Tanihara K., Matsukage A. Expression of active rat DNA polymerase beta in Escherichia coli. Biochemistry. 1988 Apr 19;27(8):2983–2990. doi: 10.1021/bi00408a048. [DOI] [PubMed] [Google Scholar]
  8. Döring T., Mitchell P., Osswald M., Bochkariov D., Brimacombe R. The decoding region of 16S RNA; a cross-linking study of the ribosomal A, P and E sites using tRNA derivatized at position 32 in the anticodon loop. EMBO J. 1994 Jun 1;13(11):2677–2685. doi: 10.1002/j.1460-2075.1994.tb06558.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gibson K. J., Benkovic S. J. Synthesis and application of derivatizable oligonucleotides. Nucleic Acids Res. 1987 Aug 25;15(16):6455–6467. doi: 10.1093/nar/15.16.6455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hanna M. M., Zhang Y., Reidling J. C., Thomas M. J., Jou J. Synthesis and characterization of a new photocrosslinking CTP analog and its use in photoaffinity labeling E. coli and T7 RNA polymerases. Nucleic Acids Res. 1993 May 11;21(9):2073–2079. doi: 10.1093/nar/21.9.2073. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hori N., Iwai S., Inoue H., Ohtsuka E. Photoaffinity labeling of T4 endonuclease V with a substrate containing a phenyldiazirine derivative. J Biol Chem. 1992 Aug 5;267(22):15591–15594. [PubMed] [Google Scholar]
  12. Kawaguchi T., Yamaguchi T., Tanaka S., Tashiro Y., Saneyoshi M. Synthetic nucleosides and nucleotides. 37. Antisense oligodeoxynucleotides bearing 5-(phenylethyl)-2'-deoxyuridylate at the 3'-terminus: exonuclease-resistant molecule with natural phosphodiester backbone. J Pharm Sci. 1996 Aug;85(8):815–818. doi: 10.1021/js9600843. [DOI] [PubMed] [Google Scholar]
  13. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  14. Lee D. K., Evans R. K., Blanco J., Gottesfeld J., Johnson J. D. Contacts between 5 S DNA and Xenopus TFIIIA identified using 5-azido-2'-deoxyuridine-substituted DNA. J Biol Chem. 1991 Sep 5;266(25):16478–16484. [PubMed] [Google Scholar]
  15. Levina A. S., Tabatadse D. R., Khalimskaya L. M., Prichodko T. A., Shishkin G. V., Alexandrova L. A., Zarytova V. P. Oligonucleotide derivatives bearing reactive and stabilizing groups attached to C5 of deoxyuridine. Bioconjug Chem. 1993 Sep-Oct;4(5):319–325. doi: 10.1021/bc00023a003. [DOI] [PubMed] [Google Scholar]
  16. Levina A. S., Tabatadze D. R., Dobrikov M. I., Shishkin G. V., Khalimskaya L. M., Zarytova V. P. Site-specific photomodification of single-stranded DNA targets by arylazide and perfluoroarylazide derivatives of oligonucleotides. Antisense Nucleic Acid Drug Dev. 1996 Summer;6(2):119–126. doi: 10.1089/oli.1.1996.6.119. [DOI] [PubMed] [Google Scholar]
  17. Levina A. S., Tabatadze D. R., Dobrikov M. I., Shishkin G. V., Zarytova V. P. Sequence-specific photomodification of single-stranded and double-stranded DNA fragments by oligonucleotide perfluoroarylazide derivative. Antisense Nucleic Acid Drug Dev. 1996 Summer;6(2):127–132. doi: 10.1089/oli.1.1996.6.127. [DOI] [PubMed] [Google Scholar]
  18. Meffert R., Dose K. UV-induced cross-linking of proteins to plasmid pBR322 containing 8-azidoadenine 2'-deoxyribonucleotides. FEBS Lett. 1988 Nov 7;239(2):190–194. doi: 10.1016/0014-5793(88)80914-1. [DOI] [PubMed] [Google Scholar]
  19. Meffert R., Rathgeber G., Schäfer H. J., Dose K. UV-induced cross-linking of Tet repressor to DNA containing tet operator sequences and 8-azidoadenines. Nucleic Acids Res. 1990 Nov 25;18(22):6633–6636. doi: 10.1093/nar/18.22.6633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mullen M. A., Wang H., Wilcox K., Herman T. Characterization of a Max:DNA complex by cross-linking to photoactive oligonucleotides. DNA Cell Biol. 1994 May;13(5):521–530. doi: 10.1089/dna.1994.13.521. [DOI] [PubMed] [Google Scholar]
  21. Muralikrishna P., Cooperman B. S. A photolabile oligodeoxyribonucleotide probe of the decoding site in the small subunit of the Escherichia coli ribosome: identification of neighboring ribosomal components. Biochemistry. 1994 Feb 15;33(6):1392–1398. doi: 10.1021/bi00172a015. [DOI] [PubMed] [Google Scholar]
  22. Osswald M., Döring T., Brimacombe R. The ribosomal neighbourhood of the central fold of tRNA: cross-links from position 47 of tRNA located at the A, P or E site. Nucleic Acids Res. 1995 Nov 25;23(22):4635–4641. doi: 10.1093/nar/23.22.4635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Yamaguchi T., Saneyoshi M. A photolabile 2',3'-dideoxyuridylate analog bearing an aryl(trifluoromethyl)diazirine moiety: photoaffinity labeling of HIV-1 reverse transcriptase. Nucleic Acids Res. 1996 Sep 1;24(17):3364–3369. doi: 10.1093/nar/24.17.3364. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Yamaguchi T., Saneyoshi M. A photolabile 2',3'-dideoxyuridylate analog bearing an aryl(trifluoromethyl)diazirine moiety: photoaffinity labeling of HIV-1 reverse transcriptase. Nucleic Acids Res. 1996 Sep 1;24(17):3364–3369. doi: 10.1093/nar/24.17.3364. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Yang S. W., Nash H. A. Specific photocrosslinking of DNA-protein complexes: identification of contacts between integration host factor and its target DNA. Proc Natl Acad Sci U S A. 1994 Dec 6;91(25):12183–12187. doi: 10.1073/pnas.91.25.12183. [DOI] [PMC free article] [PubMed] [Google Scholar]

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