Rogers Faye A, Manoharan Muthiah, Rabinovitch Peter, Ward David C, Glazer Peter M
Department of Therapeutic Radiology, Yale University School of Medicine, 295 Congress Avenue, New Haven, CT 068520, USA.
Nucleic Acids Res. 2004 Dec 15;32(22):6595-604. doi: 10.1093/nar/gkh998. Print 2004.
Triplex-forming oligonucleotides (TFOs) are DNA-binding molecules, which offer the potential to selectively modulate gene expression. However, the biological activity of TFOs as potential antigene compounds has been limited by cellular uptake. Here, we investigate the effect of cell-penetrating peptides on the biological activity of TFOs as measured in an assay for gene-targeted mutagenesis. Using the transport peptide derived from the third helix of the homeodomain of antennapedia (Antp), we tested TFO-peptide conjugates compared with unmodified TFOs. TFOs covalently linked to Antp resulted in a 20-fold increase in mutation frequency when compared with 'naked' oligonucleotides. There was no increase above background in mutation frequency when Antp by itself was added to the cells or when Antp was linked to mixed or scrambled sequence control oligonucleotides. In addition, the TFO-peptide conjugates increased the mutation frequency of the target gene, and not the control gene, in a dose-responsive manner. Confocal microscopy using labeled oligonucleotides indicated increased cellular uptake of TFOs when linked to Antp, consistent with the gene-targeting data. These results suggest that peptide conjugation may enhance intranuclear delivery of reagents designed to bind to chromosomal DNA.
三链形成寡核苷酸（TFOs）是一类DNA结合分子，具有选择性调节基因表达的潜力。然而，TFOs作为潜在的反基因化合物，其生物活性受到细胞摄取的限制。在此，我们研究了细胞穿透肽对TFOs生物活性的影响，该活性通过基因靶向诱变实验进行测定。我们使用源自触角足同源结构域第三螺旋的转运肽（Antp），将TFO-肽缀合物与未修饰的TFOs进行比较测试。与“裸露”的寡核苷酸相比，与Antp共价连接的TFOs导致突变频率增加了20倍。当单独将Antp添加到细胞中，或者将Antp与混合或随机序列的对照寡核苷酸连接时，突变频率没有高于背景值的增加。此外，TFO-肽缀合物以剂量响应方式增加了靶基因而非对照基因的突变频率。使用标记寡核苷酸的共聚焦显微镜显示，与Antp连接时TFOs的细胞摄取增加，这与基因靶向数据一致。这些结果表明，肽缀合可能增强设计用于与染色体DNA结合的试剂的核内递送。
