Potassium-resistant triple helix formation and improved intracellular gene targeting by oligodeoxyribonucleotides containing 7-deazaxanthine.
Potassium-resistant triple helix formation and improved intracellular gene targeting by oligodeoxyribonucleotides containing 7-deazaxanthine.
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通过含有 7-脱氮黄嘌呤的寡脱氧核糖核苷酸形成抗钾三螺旋并改善细胞内基因靶向。
DOI：
10.1093/nar/25.3.633
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发表时间：
1997
影响因子：
14.9
通讯作者：
Glazer,PM
中科院分区：
文献类型：
--
作者：
Faruqi,AF;Krawczyk,SH;Matteucci,MD;Glazer,PM
富含嘌呤的寡核苷酸在反平行基序中形成三螺旋被生理浓度的钾抑制。用7-脱氮黄嘌呤（c7 X）取代已被建议作为克服该效应的策略。我们已经测试了这一点，通过检查三重螺旋形成bothin体外和体内的一系列三重螺旋形成寡核苷酸（TFOs）含有鸟嘌呤加上腺嘌呤，胸腺嘧啶，或c7 X。TFOs在5′端与peptide偶联，并被设计为与upF突变报告基因的一部分结合。使用在vitrogel迁移率变动分析，我们发现，三链体形成的c7 X-取代的TFO是相对抵抗的存在下，140 mM的K+。在哺乳动物细胞的基因靶向实验中，含c7 X的TFO也是上级的，在设计用于检测由第三链定向的peptide加合物诱导的突变的基于穿梭载体的诱变测定中产生4至5倍的高突变频率。当磷酸二酯骨架被硫代磷酸酯骨架取代时，c7 X-TFOs的体外结合不受影响，但体内三螺旋形成的效率降低。这些结果表明c7 X取代在体内基因靶向实验中的实用性，并且它们表明三链体抗基因策略的可行性可以通过核苷酸化学的进步而显著增强。
Triple helix formation by purine-rich oligonucleotides in the anti-parallel motif is inhibited by physiological concentrations of potassium. Substitution with 7-deazaxanthine (c7X) has been suggested as a strategy to overcome this effect. We have tested this by examining triple helix formation bothin vitroandin vivoby a series of triple helix-forming oligonucleotides (TFOs) containing guanine plus either adenine, thymine, or c7X. The TFOs were conjugated to psoralen at the 5′ end and were designed to bind to a portion of thesupFmutation reporter gene. Usingin vitrogel mobility shift assays, we found that triplex formation by the c7X-substituted TFOs was relatively resistant to the presence of 140 mM K+. The c7X-containing TFOs were also superior in gene targeting experiments in mammalian cells, yielding 4- to 5-fold higher mutation frequencies in a shuttle vector-based mutagenesis assay designed to detect mutations induced by third strand-directed psoralen adducts. When the phosphodiester backbone was replaced by a phosphorothioate one, thein vitrobinding of the c7X-TFOs was not affected, but the efficiency ofin vivotriple helix formation was reduced. These results indicate the utility of the c7X substitution forin vivogene targeting experiments, and they show that the feasibility of the triplex anti-gene strategy can be significantly enhanced by advances in nucleotide chemistry.