Highly efficient sequence-specific DNA interstrand cross-linking by pyrrole/imidazole CPI conjugates

被引:27
作者
Bando, T
Narita, A
Saito, I
Sugiyama, H
机构
[1] Tokyo Med & Dent Univ, Inst Biomat & Bioengn, Div Biofunct Mol, Chiyoda Ku, Tokyo 1010062, Japan
[2] Japan Sci & Technol Corp, SORST, Tokyo, Japan
[3] Kyoto Univ, Fac Engn, Dept Synthet Chem & Biol Chem, Sakyo Ku, Kyoto 6068501, Japan
关键词
D O I
10.1021/ja028459b
中图分类号
O6 [化学];
学科分类号
0703 ;
摘要
We have developed a novel type of DNA interstrand cross-linking agent by synthesizing dimers of a pyrrole (Py)/imidazole (Im)-diamide-CPI conjugate, ImPyLDu86 (1), connected using seven different linkers. The tetramethylene linker compound, 7b, efficiently produces DNA interstrand cross-links at the nine-base-pair sequence, 5'-PyGGC(T/A)GCCPu-3', only in the presence of a partner triamide, ImImPy. For efficient cross-linking by 7b with ImImPy, one A-T base pair between two recognition sites was required to accommodate the linker region. Elimination of the A-T base pair and insertion of an additional A-T base pair and substitution with a G.C base pair significantly reduced the degree of cross-linking. The sequence specificity of the interstrand cross-linking by 7b was also examined in the presence of various triamides. The presence of ImImIm slightly reduced the formation of a cross-linked product compared to ImImPy. The mismatch partners, ImPyPy and PyImPy, did not produce an interstrand cross-link product with 7b, whereas ImPyPy and PyImPy induced efficient alkylation at their matching site with 7b. The interstrand cross-linking abilities of 7b were further examined using denaturing polyacrylamide gel electrophoresis with 5'-Texas Red-labeled 400- and 67-bp DNA fragments. The sequencing gel analysis of the 400-bp DNA fragment with ImImPy demonstrated that 7b alkylates several sites on the top and bottom strands, including one interstrand cross-linking match site, 5'-PyGGC(T/A)GCCPu-3'. To obtain direct evidence of interstrand cross-linkages on longer DNA fragments, a simple method using biotin-labeled complementary strands was developed, which produced a band corresponding to the interstrand cross-linked site on both top and bottom strands. Densitometric analysis indicated that the contribution of the interstrand cross-link in the observed alkylation bands was approximately 40%. This compound efficiently cross-linked both strands at the target sequence. The present system consisted of a 1:2 complex of the alkylating agent and its partner ImImPy and caused an interstrand cross-linking in a sequence-specific fashion according to the base-pair recognition rule of Py-Im polyamides.
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页码:3471 / 3485
页数:15
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