Torigoe H, Ferdous A, Watanabe H, Akaike T, Maruyama A
Gene Bank, Tsukuba Life Science Center, The Institute of Physical and Chemical Research (RIKEN), 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan.
J Biol Chem. 1999 Mar 5;274(10):6161-7. doi: 10.1074/jbc.274.10.6161.
Extreme instability of pyrimidine motif triplex DNA at physiological pH severely limits its use for artificial control of gene expression in vivo. Stabilization of the pyrimidine motif triplex at physiological pH is therefore of great importance in improving its therapeutic potential. To this end, isothermal titration calorimetry interaction analysis system and electrophoretic mobility shift assay have been used to explore the thermodynamic and kinetic effects of our previously reported triplex stabilizer, poly (L-lysine)-graft-dextran (PLL-g-Dex) copolymer, on pyrimidine motif triplex formation at physiological pH. Both the thermodynamic and kinetic analyses have clearly indicated that in the presence of the PLL-g-Dex copolymer, the binding constant of the pyrimidine motif triplex formation at physiological pH was about 100 times higher than that observed without any triplex stabilizer. Of importance, the triplex-promoting efficiency of the copolymer was more than 20 times higher than that of physiological concentrations of spermine, a putative intracellular triplex stabilizer. Kinetic data have also demonstrated that the observed copolymer-mediated promotion of the triplex formation at physiological pH resulted from the considerable increase in the association rate constant rather than the decrease in the dissociation rate constant. Our results certainly support the idea that the PLL-g-Dex copolymer could be a key material and may eventually lead to progress in therapeutic applications of the antigene strategy in vivo.
嘧啶基序三链体DNA在生理pH值下的极端不稳定性严重限制了其在体内人工控制基因表达中的应用。因此，在生理pH值下稳定嘧啶基序三链体对于提高其治疗潜力至关重要。为此，已使用等温滴定量热法相互作用分析系统和电泳迁移率变动分析来探究我们先前报道的三链体稳定剂聚（L-赖氨酸）-接枝-葡聚糖（PLL-g-Dex）共聚物在生理pH值下对嘧啶基序三链体形成的热力学和动力学影响。热力学和动力学分析均清楚表明，在存在PLL-g-Dex共聚物的情况下，生理pH值下嘧啶基序三链体形成的结合常数比未使用任何三链体稳定剂时观察到的结合常数高约100倍。重要的是，该共聚物促进三链体形成的效率比假定的细胞内三链体稳定剂精胺的生理浓度促进三链体形成的效率高20倍以上。动力学数据还表明，在生理pH值下观察到的共聚物介导的三链体形成促进作用是由于缔合速率常数的显著增加而非解离速率常数的降低。我们的结果无疑支持了PLL-g-Dex共聚物可能是关键材料这一观点，并且最终可能会推动反基因策略在体内治疗应用方面取得进展。
