Lin F L, Majumdar A, Klotz L C, Reszka A P, Neidle S, Seidman M M
Laboratory of Molecular Genetics, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224, USA.
J Biol Chem. 2000 Dec 15;275(50):39117-24. doi: 10.1074/jbc.M005404200.
Triple helix-forming oligonucleotides may be useful as gene-targeting reagents in vivo, for applications such as gene knockout. One important property of these complexes is their often remarkable stability, as demonstrated in solution and in cells following transfection. Although encouraging, these measurements do not necessarily report triplex stability in cellular compartments that support DNA functions such as replication and mutagenesis. We have devised a shuttle vector plasmid assay that reports the stability of triplexes on DNA that undergoes replication and mutagenesis. The assay is based on plasmids with novel variant supF tRNA genes containing embedded sequences for triplex formation and psoralen cross-linking. Triple helix-forming oligonucleotides were linked to psoralen and used to form triplexes on the plasmids. At various times after introduction into cells, the psoralen was activated by exposure to long wave ultraviolet light (UVA). After time for replication and mutagenesis, progeny plasmids were recovered and the frequency of plasmids with mutations in the supF gene determined. Site-specific mutagenesis by psoralen cross-links was dependent on precise placement of the psoralen by the triple helix-forming oligonucleotide at the time of UVA treatment. The results indicated that both pyrimidine and purine motif triplexes were much less stable on replicated DNA than on DNA in vitro or in total transfected DNA. Incubation of cells with amidoanthraquinone-based triplex stabilizing compounds enhanced the stability of the pyrimidine triplex.
三链螺旋形成寡核苷酸在体内可能作为基因靶向试剂用于诸如基因敲除等应用。这些复合物的一个重要特性是它们通常具有显著的稳定性，这在转染后的溶液和细胞中得到了证明。尽管令人鼓舞，但这些测量结果不一定反映在支持DNA功能（如复制和诱变）的细胞区室中的三链体稳定性。我们设计了一种穿梭载体质粒测定法，用于报告在经历复制和诱变的DNA上三链体的稳定性。该测定法基于带有新型变体supF tRNA基因的质粒，该基因包含用于三链体形成和补骨脂素交联的嵌入序列。三链螺旋形成寡核苷酸与补骨脂素连接，并用于在质粒上形成三链体。在引入细胞后的不同时间，通过暴露于长波紫外线（UVA）激活补骨脂素。在经过复制和诱变的时间后，回收子代质粒并确定supF基因中带有突变的质粒频率。补骨脂素交联引起的位点特异性诱变取决于在UVA处理时三链螺旋形成寡核苷酸对补骨脂素的精确定位。结果表明，嘧啶和嘌呤基序三链体在复制的DNA上的稳定性远低于在体外DNA或总转染DNA上的稳定性。用基于氨基蒽醌的三链体稳定化合物孵育细胞可增强嘧啶三链体的稳定性。
