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首页> 外文期刊>The FEBS journal >Monitoring DNA triplex formation using multicolorfluorescence and application to insulin-like growth factor Ipromoter downregulation
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Monitoring DNA triplex formation using multicolorfluorescence and application to insulin-like growth factor Ipromoter downregulation

机译：使用多色荧光监测DNA三链体的形成并将其应用于胰岛素样生长因子I启动子的下调

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摘要

Inhibition of insulin-like growth factor I (IGF–I) signaling is a promisingantitumor strategy and nucleic acid-based approaches have been investigatedto target genes in the pathway. Here, we sought to modulate IGF-Itranscriptional activity using triple helix formation. The IGF-I P1 promotercontains a purine/pyrimidine (R/Y) sequence that is pivotal for transcriptionas determined by deletion analysis and can be targeted with a triplex-formingoligonucleotide (TFO). We designed modified purine- and pyrimidinerichTFOs to bind to the R/Y sequence. To monitor TFO binding, wedeveloped a fluorescence-based gel-retardation assay that allowed independentdetection of each strand in three-stranded complexes using end-labelingwith Alexa 488, cyanine (Cy)3 and Cy5 fluorochromes. We characterizedTFOs for their ability to inhibit restriction enzyme activity, compete withDNA-binding proteins and inhibit IGF-I transcription in reporter assays.TFOs containing modified nucleobases, 5-methyl-2′-deoxycytidine and 5-propynyl-2′-deoxyuridine, specifically inhibited restriction enzyme cleavageand formed triplexes on the P1 promoter fragment. In cells, deletion of theR/Y-rich sequence led to 48% transcriptional inhibition of a reporter gene.Transfection with TFOs inhibited reporter gene activity to a similar extent,whereas transcription from a mutant construct with an interrupted R/Yregion was unaffected, strongly suggesting the involvement of triplex formationin the inhibitory mechanisms. Our results indicate that nuclease-resistantTFOs will likely inhibit endogenous IGF-I gene function in cells.
机译：抑制胰岛素样生长因子I（IGF–I）信号传导是一种有前途的抗肿瘤策略，并且已经研究了基于核酸的方法来靶向该途径中的基因。在这里，我们试图使用三重螺旋的形成来调节IGF-转录活性。 IGF-1P1启动子包含嘌呤/嘧啶（R / Y）序列，其通过缺失分析确定对于转录是关键的，并且可以用三链体形成寡核苷酸（TFO）靶向。我们设计了修饰的富含嘌呤和嘧啶的TFO以结合到R / Y序列。为了监测TFO结合，我们开发了一种基于荧光的凝胶延迟测定法，该方法可使用Alexa 488，花青（Cy）3和Cy5荧光染料末端标记，独立检测三链复合物中的每条链。我们对TFO的抑制酶活性，与DNA结合蛋白竞争和抑制IGF-I转录的能力进行了表征.TFO包含修饰的核碱基，5-甲基-2'-脱氧胞苷和5-丙炔基-2'-脱氧尿苷抑制限制酶的切割并在P1启动子片段上形成三链体。在细胞中，富含R / Y序列的缺失导致报告基因的转录抑制48％。用TFOs转染可以类似程度地抑制报告基因的活性，而突变的构建体中具有中断的R / Y区域的转录却不受影响，强烈提示三链体的形成与抑制机制有关。我们的结果表明，耐核酸酶的TFO可能会抑制细胞中内源性IGF-1基因的功能。

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