Degols G, Clarenc J P, Lebleu B, Léonetti J P
Institut de Génétique Móléculaire du CNRS, Montpellier, France.
J Biol Chem. 1994 Jun 17;269(24):16933-7.
Triple helix formation of nucleic acids is the most rational approach to designing site-specific transcription inhibitors. To increase their efficiency, reactive moieties such as psoralen or ethenocytosine have been introduced on the third strand. In transfected cells, these compounds induce a site-specific covalent binding of the third strand to the targeted sequence and efficiently block RNA polymerases. However, the stability of this transcription inhibition has never been checked. We have designed a plasmid containing a triple helix binding site in the coding region of the beta-galactosidase reporter gene and a polymerase chain reaction assay to follow quantitatively the cross-link of a psoralen-derivatized third strand in transfected cells. This assay has revealed that the cross-link was removed within a few hours, leading only to a transitory inhibition of gene expression. Control experiments in DNA repair-deficient cells suggest the implication of repair enzymes in this process.
核酸三链体的形成是设计位点特异性转录抑制剂最合理的方法。为提高其效率，已在第三条链上引入了诸如补骨脂素或乙烯胞嘧啶等反应性基团。在转染细胞中，这些化合物可诱导第三条链与靶向序列发生位点特异性共价结合，并有效阻断RNA聚合酶。然而，这种转录抑制的稳定性从未得到检测。我们设计了一种质粒，其在β-半乳糖苷酶报告基因的编码区含有一个三链体结合位点，并设计了一种聚合酶链反应检测方法，以定量追踪转染细胞中补骨脂素衍生的第三条链的交联情况。该检测方法显示，交联在数小时内就被去除，仅导致基因表达的短暂抑制。在DNA修复缺陷细胞中进行的对照实验表明修复酶参与了这一过程。
