Ferdous A, Akaike T, Maruyama A
Department of Biomolecular Engineering, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan.
Bioconjug Chem. 2000 Jul-Aug;11(4):520-6. doi: 10.1021/bc990166t.
We previously reported a novel strategy to stabilize purine motif triplex DNA within a mammalian gene promoter at physiologically relevant pH, temperature, and potassium (K(+)) concentrations by a comb-type poly(L-lysine)-graft-dextran copolymer [Ferdous et al., (1998) Nucleic Acids Res. 26, 3949-3954]. Here we describe the major contribution(s) of the copolymer to stabilize the purine motif triplex DNA at physiological K(+) concentrations. Self-aggregation through guanine-quartet formation of guanine-rich (G-rich) triplex-forming oligonucleotides (TFOs) has long been proposed for K(+)-mediated inhibition of the purine motif triplex formation. However, this was not the case for the severe inhibitory effect of K(+) observed under our reaction conditions. Rather significant decrease in rate of triplex formation involving a G-rich TFO was a major factor to confer K(+) inhibition. Interestingly, in the presence of the copolymer the rate of triplex formation was tremendously increased and K(+)-induced dissociation of preformed triplexes was not observed. Moreover, the triplex-promoting/stabilizing efficiency of the copolymer was amazingly higher than that of physiological concentrations of spermine. An absolute increase in binding constant of the TFO to the target duplex could therefore be the predominant mechanistic source for the copolymer-mediated triplex stabilization under physiological conditions in vitro.
我们之前报道了一种新策略，通过梳型聚（L-赖氨酸）-接枝-葡聚糖共聚物在生理相关的pH、温度和钾（K⁺）浓度下稳定哺乳动物基因启动子内的嘌呤基序三链体DNA[费尔多斯等人，（1998年）《核酸研究》26，3949 - 3954]。在此我们描述了该共聚物在生理K⁺浓度下对稳定嘌呤基序三链体DNA的主要贡献。长期以来，人们一直认为富含鸟嘌呤（G-rich）的三链体形成寡核苷酸（TFOs）通过鸟嘌呤四重体形成的自聚集作用介导K⁺对嘌呤基序三链体形成的抑制。然而，在我们的反应条件下观察到的K⁺的严重抑制作用并非如此。涉及富含G的TFO的三链体形成速率的显著降低是导致K⁺抑制的主要因素。有趣的是，在共聚物存在下，三链体形成速率大幅增加，并且未观察到K⁺诱导的预先形成的三链体的解离。此外，共聚物促进/稳定三链体的效率惊人地高于生理浓度的精胺。因此，在体外生理条件下，TFO与靶双链体结合常数的绝对增加可能是共聚物介导的三链体稳定的主要机制来源。
