High-affinity triple helix formation by synthetic oligonucleotides at a site within a selectable mammalian gene.
High-affinity triple helix formation by synthetic oligonucleotides at a site within a selectable mammalian gene.
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通过合成寡核苷酸在可选择的哺乳动物基因内的位点形成高亲和力三螺旋。
DOI：
10.1021/bi00021a040
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发表时间：
1995
期刊：
影响因子：
2.9
通讯作者：
Wilson,JH
中科院分区：
文献类型：
--
作者：
Vasquez,KM;Wensel,TG;Hogan,ME;Wilson,JH
1995年3月24日接收的修订版Mandalpt ®摘要：单链寡核苷酸通过形成三链体DNA特异性识别双链体DNA是靶向基因组特定区域的合理方法。通过筛选哺乳动物基因内允许在细胞培养中进行遗传选择的三螺旋形成的一些潜在靶位点，我们已经确定了仓鼠腺嘌呤磷酸核糖基转移酶（APRT）基因的内含子1内的一个位点，该位点特异性地以高亲和力结合三链体形成寡脱氧核糖核苷酸（TFO）。在三链体形成的最佳条件下，平衡解离常数在纳摩尔范围内（Kd= 7 × 10 - 1 M）。这种高亲和性结合是非常特异的，因为105倍过量的基因组DNA减少三链体形成不到10倍，并且在携带APRT基因的6928-bp质粒内，仅发现含有内含子1位点的限制性片段结合TFO。DNA酶I保护测定的结果与TFO通过双链体大沟中的反向Hoogsteen氢键以反平行方向结合一致。我们研究了三链体形成的动力学以及TFO的离子组成和化学修饰对三链体形成的影响。虽然三链体的形成不需要二价阳离子，但Mg 2+明显通过抑制TFO解离来稳定三链体，在Mg 2+浓度高于5 mM时，三链体的平均结合寿命> 17 h。抑制三链体形成的顺序为K+> Li+> Na+。6-硫代鸟苷（6-TG）修饰的TFO的结合对K+的敏感性比未修饰的TFO的结合低得多，这与6-TG抑制G-四联体形成（一种可能的K+依赖性竞争反应）一致。最后，在与双链体中的胞嘧啶碱基相互作用的位置处含有胸腺嘧啶的TFO形成三链体，其亲和力类似于含有咪唑衍生物或5-氟尿嘧啶代替胸腺嘧啶的TFO的亲和力。
Revised Manuscript Received March 24, 1995® abstract: Specific recognition of duplex DNA by a single-stranded oligonucleotide via the formation of triplex DNA is a rational approach for targeting specific regions of a genome. By screening a number of potential target sites for triple helix formation within mammalian genes that allow genetic selection in cell culture, we have identified a site within intron 1 of the hamster adenine phosphoribosyltransferase (APRT) gene that specifically binds a triplex-forming oligodeoxyribonucleotide (TFO) with high affinity. Under optimal conditions for triplex formation, the equilibrium dissociation constant is in the nanomolar range (Kd= 7 x 10_1 M). This high-affinity binding is very specific, as a 105-fold excess of genomicDNA reduced triplex formation less than 10-fold, and within a 6928-bp plasmid bearing the APRT gene, only restriction fragments containing the intron 1 site were found to bind the TFO. Results of DNase I protection assays were consistent with the TFO binding in an antiparallel orientation via reverse Hoogsteen hydrogen bonds in the major groove of the duplex. We have examined the kinetics of triplex formation as well as the effects of ioniccomposition and chemical modifications of the TFO on triplex formation. While divalent cations were not required for triplexformation, Mg2+ stabilized the triplex apparently through inhibitionof TFO dissociation, with a mean bound lifetime of> 17 h for the triplex at Mg2+ concentrations above 5 mM. Monovalent cationshad little or no effect on triplex formation at low concentrations (10 mM) but had a negative effect at higher concentrations, with inhibition of triplex formation in the order K+» Li+> Na+. Binding of a 6-thioguanosine (6-TG) modified TFO was much less sensitive to K+ than binding of the unmodified TFO, consistent with 6-TG inhibition of G-quartet formation, a likely K+-dependent competing reaction. Finally, the TFO containing thymines at positions interacting with cytosine bases in the duplex formed triplex with an affinity similar to those of TFOs containing either an imidazole derivative or 5-fluorouracil in place of thymine.