Sequence-specific protection of plasmid DNA from restriction endonuclease hydrolysis by pyrrole–imidazole–cyclopropapyrroloindole conjugates

摘要

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 µM 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.