Kuramochi Hidetoshi, Yonezawa Yasuo
Research Center for Material Cycles and Waste Management, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan.
J Biosci Bioeng. 2003;95(3):225-30.
To investigate the effects of ATP, Mg2+, and a mixture of the two on the formation of large DNA aggregates induced by spermidine, we constructed novel phase diagrams of the spermidine/DNA/Tris-buffer system in the presence of these compounds. These diagrams reveal not only the difference in the inhibition of the aggregate formation between ATP and Mg2+ but also a type of buffering effect whereby the complex formation of ATP and Mg2+ diminishes their individual inhibitory actions. Furthermore, the equilibrium composition of ATP, spermidine, Mg2+, and their complexes in the liquid phase was estimated using the equilibrium constants for the complex formations and their mass balances. From this solution equilibrium analysis, the threshold condition for DNA aggregation is suggested. The phase diagrams and equilibrium analysis presented here are useful to predict the threshold of aggregation in a system similar to in vitro transcription. Finally, we recommend that region b in this paper, where the variation in the concentration of either ATP or Mg2+ had little effect on the DNA aggregation, should be used to establish the initial conditions of in vitro RNA synthesis.
为了研究ATP、Mg2+以及二者混合物对亚精胺诱导的大DNA聚集体形成的影响，我们构建了在这些化合物存在下的亚精胺/DNA/Tris缓冲液体系的新型相图。这些相图不仅揭示了ATP和Mg2+在抑制聚集体形成方面的差异，还揭示了一种缓冲效应，即ATP与Mg2+的络合作用减弱了它们各自的抑制作用。此外，利用络合物形成的平衡常数及其质量平衡，估算了液相中ATP、亚精胺、Mg2+及其络合物的平衡组成。通过这种溶液平衡分析，提出了DNA聚集的阈值条件。本文给出的相图和平衡分析有助于预测类似于体外转录体系中的聚集阈值。最后，我们建议本文中的区域b可用于建立体外RNA合成的初始条件，在该区域中，ATP或Mg2+浓度的变化对DNA聚集影响很小。
