Sequence-specific protection of plasmid DNA from restriction endonuclease hydrolysis by pyrrole-imidazole-cyclopropapyrroloindole conjugates.
The pyrrole-imidazole (Py-Im) triamide-cyclopropa pyrroloindole (CPI) conjugates ImPyImLDu86 (7) and ImImPyLDu86 (14) were synthesized and their alkylating activities and inhibitory effects on DNA hydrolysis by restriction endonucleases were examined. Sequencing gel analysis demonstrated that conjugates 7 and 14 specifically alkylated DNA at 5'-CGCGCG-3' and 5'-PyGGCCPu-3', respectively. Agarose gel electrophoresis indicated that incubation of a supercoiled plasmid, pSPORT I (4109 bp), with conjugate 7 effectively inhibited its hydrolysis by BssHII (5'-G_CGCGC-3'), whereas conjugate 14 had no effect on this hydrolysis. These results suggest that conjugate 7 sequence-specifically inhibits the hydrolysis of DNA by BssHII. Sequence-specific alkylation by the Py-Im triamide-CPI conjugates was further confirmed by inhibition of the Eco52I (5'-C_GGCCG-3') hydrolysis of conjugate 14-treated pQBI PGK (5387 bp). In clear contrast, hydrolysis of pQB1 PGK by DraI (3'-TTT_AAA-3') was not inhibited by 5 micro M conjugate 14. That ImImPy did not inhibit the hydrolysis of pQB1 PGK indicates that covalent bond formation is necessary for inhibition. A similar experiment, using linear pQBI PGK, achieved the same extent of protection of the DNA with approximately half the concentration of conjugate 14 as was required to protect supercoiled DNA from hydrolysis.
Division of Biofunctional Molecules, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Surugadai, Kanda, Chiyoda, Tokyo 101-0062, Japan.
Kazuhisa
K
Fujimoto
Hirokazu
H
Iida
Masako
M
Kawakami
Toshikazu
T
Bando
Zhi-Fu
ZF
Tao
Hiroshi
H
Sugiyama
D000478:Alkylation; D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D004278:DNA, Superhelical / Q000737:chemistry / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007093:Imidazoles / Q000737:chemistry*; D010957:Plasmids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011758:Pyrroles / Q000737:chemistry*; D017422:Sequence Analysis, DNA
D004278:DNA, Superhelical; D007093:Imidazoles; D011758:Pyrroles; D004247:DNA; D004262:DNA Restriction Enzymes; C044171:endodeoxyribonuclease BSSHII; C046751:endodeoxyribonuclease XmaIII; D015252:Deoxyribonucleases, Type II Site-Specific; C035594:TTTAAA -specific type II deoxyribonucleases