Hampel K J, Crosson P, Lee J S
Department of Biochemistry, University of Saskatchewan, Saskatoon, Canada.
Biochemistry. 1991 May 7;30(18):4455-9. doi: 10.1021/bi00232a012.
The stability of triplex DNA was investigated in the presence of the polyamines spermine and spermidine by four different techniques. First, thermal-denaturation analysis of poly[d(TC)].poly[d(GA)] showed that at low ionic strength and pH 7, 3 microM spermine was sufficient to cause dismutation of all of the duplex to the triplex conformation. A 10-fold higher concentration of spermidine produced a similar effect. Second, the kinetics of the dismutation were measured at pH 5 in 0.2 M NaCl. The addition of 500 microM spermine increased the rate by at least 2-fold. Third, in 0.2 M NaCl, the mid-point of the duplex-to-triplex dismutation occurred at a pH of 5.8, but this was increased by nearly one pH unit in the presence of 500 microM spermine. Fourth, intermolecular triplexes can also form in plasmids that contain purine.pyrimidine inserts by the addition of a single-stranded pyrimidine. This was readily demonstrated at pH 7.2 and 25 mM ionic strength in the presence of 100 microM spermine or spermidine. In 0.2 M NaCl, however, 1 mM polyamine is required. Since, in the eucaryotic nucleus, the polyamine concentration is in the millimolar range, then appropriate purine-pyrimidine DNA sequences may favor the triplex conformation in vivo.
通过四种不同技术研究了在多胺精胺和亚精胺存在下三链体DNA的稳定性。首先，对聚[d(TC)]·聚[d(GA)]进行热变性分析表明，在低离子强度和pH 7条件下，3 μM精胺足以使所有双链体转变为三链体构象。亚精胺浓度高10倍时会产生类似效果。其次，在pH 5的0.2 M NaCl中测量转变动力学。加入500 μM精胺使速率至少提高2倍。第三，在0.2 M NaCl中，双链体到三链体转变的中点出现在pH 5.8处，但在500 μM精胺存在下，这一数值增加了近一个pH单位。第四，通过添加单链嘧啶，在含有嘌呤·嘧啶插入片段的质粒中也能形成分子间三链体。在pH 7.2和25 mM离子强度下，100 μM精胺或亚精胺存在时很容易证明这一点。然而，在0.2 M NaCl中，则需要1 mM多胺。由于在真核细胞核中，多胺浓度处于毫摩尔范围内，那么合适的嘌呤 - 嘧啶DNA序列在体内可能有利于三链体构象的形成。
