Vasquez K M, Wensel T G, Hogan M E, Wilson J H
Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030, USA.
Biochemistry. 1995 May 30;34(21):7243-51. doi: 10.1021/bi00021a040.
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(-10) M). This high-affinity binding is very specific, as a 10(5)-fold excess of genomic DNA 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 ionic composition and chemical modifications of the TFO on triplex formation. While divalent cations were not required for triplex formation, Mg2+ stabilized the triplex apparently through inhibition of TFO dissociation, with a mean bound lifetime of > 17 h for the triplex at Mg2+ concentrations above 5 mM.(ABSTRACT TRUNCATED AT 250 WORDS)
通过形成三链体DNA，单链寡核苷酸对双链DNA进行特异性识别是靶向基因组特定区域的一种合理方法。通过在允许细胞培养中进行遗传选择的哺乳动物基因内筛选多个潜在的三链体形成靶位点，我们在仓鼠腺嘌呤磷酸核糖转移酶（APRT）基因的内含子1中鉴定出一个位点，该位点能以高亲和力特异性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，平衡解离常数处于纳摩尔范围（Kd = 7×10⁻¹⁰ M）。这种高亲和力结合非常特异，因为过量10⁵倍的基因组DNA使三链体形成减少不到10倍，并且在携带APRT基因的6928 bp质粒中，仅发现含有内含子1位点的限制性片段能结合TFO。DNase I保护试验结果与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。虽然三链体形成不需要二价阳离子，但Mg²⁺显然通过抑制TFO解离来稳定三链体，在Mg²⁺浓度高于5 mM时，三链体的平均结合寿命> 17小时。（摘要截短于250字）
