Nuclear localization of pyrrole-imidazole polyamide-fluorescein conjugates in cell culture
Nuclear localization of pyrrole-imidazole polyamide-fluorescein conjugates in cell culture
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DOI：
10.1073/pnas.2035074100
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发表时间：
2003-10-14
影响因子：
11.1
通讯作者：
Dervan, PB
中科院分区：
文献类型：
--
作者：
Best, TP;Edelson, BS;Dervan, PB
合成了一系列发夹状吡咯-咪唑聚酰胺-荧光素缀合物，并测定了其细胞定位。用5 μ M聚酰胺-荧光素偶联物处理13个细胞系，代表11种人类癌症，一种人类转化的肾细胞系，和一种鼠白血病细胞系10-14小时，然后通过共聚焦激光扫描显微镜成像。在聚酰胺部分的C末端含有β-丙氨酸残基的缀合物显示没有核定位，而缺乏β-丙氨酸残基的类似化合物强烈定位于所有测试的细胞系的核中。其他几种共轭物的定位概况表明，吡咯-咪唑序列和内容，染料的选择和位置，接头的组成，和分子量的核定位的决定因素。将荧光素连接到发夹聚酰胺的C末端，导致DNA结合亲和力降低约10倍，而结合特异性没有损失，参照错配结合位点。
A series of hairpin pyrrole-imidazole polyamide-fluorescein conjugates were synthesized and assayed for cellular localization. Thirteen cell lines, representing 11 human cancers, one human transformed kidney cell line, and one murine leukemia cell line, were treated with 5 muM polyamide-fluorescein conjugates for 10-14 h, then imaged by confocal laser scanning microscopy. A conjugate containing a beta-alanine residue at the C terminus of the polyamide moiety showed no nuclear localization, whereas an analogous compound lacking the beta-alanine residue was strongly localized in the nuclei of all cell lines tested. The localization profiles of several other conjugates suggest that pyrrole-imidazole sequence and content, dye choice and position, linker composition, and molecular weight are determinants of nuclear localization. The attachment of fluorescein to the C terminus of a hairpin polyamide results in an approximate to10-fold reduction in DNA-binding affinity, with no loss of binding specificity with reference to mismatch binding sites.