Lacoste J, François J C, Hélène C
Laboratoire de Biophysique, Muséum National d'Histoire Naturelle, INSERM Unité 201-CNRS UA 481, 43 rue Cuvier, 75231 Paris Cedex 05, France.
Nucleic Acids Res. 1997 May 15;25(10):1991-8. doi: 10.1093/nar/25.10.1991.
Purine-rich (GA)- and (GT)-containing oligophosphorothioates were investigated for their triplex-forming potential on a 23 bp DNA duplex target. In our system, GA-containing oligophosphorothioates (23mer GA-PS) were capable of triplex formation with binding affinities lower than (GA)-containing oligophosphodiesters (23mer GA-PO). The orientation of the third strand 23mers GA-PS and GA-PO was antiparallel to the purine strand of the duplex DNA target. In contrast, (GT)-containing oligophosphorothioates (23mer GT-PS) did not support triplex formation in either orientation, whereas the 23mer GT-PO oligophosphodiester demonstrated triplex formation in the antiparallel orientation. GA-PS oligonucleotides, in contrast to GT-PS oligonucleotides, were capable of self-association, but these self-associated structures exhibited lower stabilities than those formed with GA-PO oligonucleotides, suggesting that homoduplex formation (previously described for the 23mer GA-PO sequence by Noonberg et al.) could not fully account for the decrease in triplex stability when phosphorothioate linkages were used. The 23mer GA-PS oligonucleotide was covalently linked via its 5'-end to an acridine derivative (23mer Acr-GA-PS). In the presence of potassium cations, this conjugate demonstrated triplex formation with higher binding affinity than the unmodified 23mer GA-PS oligonucleotide and even than the 23mer GA-PO oligonucleotide. A (GA)-containing oligophosphodiester with two phosphorothioate linkages at both the 5'- and 3'-ends exhibited similar binding affinity to duplex DNA compared with the unmodified GA-PO oligophosphodiester. This capped oligonucleotide was more resistant to nucleases than the GA-PO oligomer and thus represents a good alternative for ex vivo applications of (GA)-containing, triplex-forming oligonucleotides, allowing a higher binding affinity for its duplex target without rapid cellular degradation.
研究了富含嘌呤的（GA）-和含（GT）的寡聚硫代磷酸酯在23bp DNA双链靶标上形成三链体的潜力。在我们的系统中，含GA的寡聚硫代磷酸酯（23聚体GA-PS）能够形成三链体，但其结合亲和力低于含（GA）的寡聚磷酸二酯（23聚体GA-PO）。第三条链23聚体GA-PS和GA-PO的方向与双链DNA靶标的嘌呤链反平行。相比之下，含（GT）的寡聚硫代磷酸酯（23聚体GT-PS）在任何一种方向上都不支持三链体形成，而23聚体GT-PO寡聚磷酸二酯在反平行方向上显示出三链体形成。与GT-PS寡核苷酸相比，GA-PS寡核苷酸能够自缔合，但这些自缔合结构的稳定性低于由GA-PO寡核苷酸形成的结构，这表明同型双链体形成（Noonberg等人先前对23聚体GA-PO序列的描述）不能完全解释使用硫代磷酸酯键时三链体稳定性的降低。23聚体GA-PS寡核苷酸通过其5'-末端与吖啶衍生物共价连接（23聚体Acr-GA-PS）。在钾离子存在下，这种缀合物显示出比未修饰的23聚体GA-PS寡核苷酸甚至比23聚体GA-PO寡核苷酸更高的结合亲和力形成三链体。与未修饰的GA-PO寡聚磷酸二酯相比，在5'-和3'-末端都具有两个硫代磷酸酯键的含（GA）的寡聚磷酸二酯对双链DNA表现出相似的结合亲和力。这种封端的寡核苷酸比GA-PO寡聚物对核酸酶更具抗性，因此代表了含（GA）的、形成三链体的寡核苷酸体外应用的良好替代品，允许对其双链靶标具有更高的结合亲和力而不会在细胞中快速降解。
