Lin S B, Kao C F, Lee S C, Kan L S
Department of Clinical Pathology, College of Medicine, National Taiwan University, Taipei, ROC.
Anticancer Drug Des. 1994 Feb;9(1):1-8.
Evidence from UV spectroscopic melting experiments indicated that the DNA oligonucleotide 5'-d-A-(G-A)7-G-3' (1), a repeating AG sequence found in the human genome, and its complement 5'-d-mC-(T-mC)7-T-3' (mC: 5-methyl-C) (2), can form both a triplex (with a Tm = 44 degrees C) and a duplex (with a Tm = 69 degrees C) around physiological pH (7.2) in micromolar concentration solution with 0.1 M NaCl. In addition, the triplex can be detected at a pH as high as 8.4 (Tm = 27 degrees C). The stability of the triplex formed by 1 and 2, as monitored by UV melting experiments, is found to increase as the pH is lowered from 8.4 (Tm = 27 degrees C) to 6 (Tm = 79 degrees C). However, the stability of the duplex, formed by 1 and 2, is found to be unchanged (the Tm is approximately 69 degrees C) in the same pH range. There is no triplex, as observed by UV with the oligonucleotides having identical sequences, when the regular cytosine base was used [d-C-(T-C)7-T, (3)] at a pH > 6 in the same concentration range. The stoichiometric ratio of the triplex formed with 1 and 2 is also found to be 1:2 by means of a UV mixing titration study. This result suggests that the conformation of the triplex of 1 and 2 also involves T.A.T and mC+.G.mC base triads. The formation of a triplex by 1 and 2 can be observed by native gel electrophoresis in submicromolar conditions with magnesium ion present. The results of this study strongly support the theory that replacement of regular cytidine nucleotides by 5-methylcytosine nucleotides facilitates the formation of the DNA triplex at physiological pH. This could thus be used an an anti-gene probe via the formation of triplex under the conditions described.
紫外光谱熔解实验的证据表明，DNA寡核苷酸5'-d-A-(G-A)7-G-3'（1）是人类基因组中发现的一种重复AG序列，及其互补链5'-d-mC-(T-mC)7-T-3'（mC：5-甲基胞嘧啶）（2），在含有0.1 M NaCl的微摩尔浓度溶液中，围绕生理pH值（7.2）时，既能形成三链体（熔解温度Tm = 44℃），也能形成双链体（熔解温度Tm = 69℃）。此外，在pH高达8.4（Tm = 27℃）时也能检测到三链体。通过紫外熔解实验监测发现，由1和2形成的三链体的稳定性随着pH从8.4（Tm = 27℃）降低到6（Tm = 79℃）而增加。然而，在相同的pH范围内，由1和2形成的双链体的稳定性保持不变（熔解温度Tm约为69℃）。当在相同浓度范围内pH > 6时，使用常规胞嘧啶碱基[d-C-(T-C)7-T，（3）]的寡核苷酸通过紫外观察不到三链体。通过紫外混合滴定研究还发现，由1和2形成的三链体的化学计量比也为1:2。该结果表明，1和2的三链体构象也涉及T.A.T和mC+.G.mC碱基三联体。在存在镁离子的亚微摩尔条件下，通过非变性凝胶电泳可以观察到1和2形成三链体。本研究结果有力地支持了以下理论：用5-甲基胞嘧啶核苷酸取代常规胞嘧啶核苷酸有助于在生理pH值下形成DNA三链体。因此，在所述条件下通过形成三链体，这可被用作反基因探针。
