TRIPLE HELIX FORMATION BY OLIGOPURINE-OLIGOPYRIMIDINE DNA FRAGMENTS - ELECTROPHORETIC AND THERMODYNAMIC BEHAVIOR
TRIPLE HELIX FORMATION BY OLIGOPURINE-OLIGOPYRIMIDINE DNA FRAGMENTS - ELECTROPHORETIC AND THERMODYNAMIC BEHAVIOR
复制标题
DOI：
10.1016/s0022-2836(05)80267-0
复制
发表时间：
1990-06-20
影响因子：
5.6
通讯作者：
VANBOOM, JH
中科院分区：
文献类型：
--
作者：
MANZINI, G;XODO, LE;VANBOOM, JH
26聚寡脱氧核苷酸d（GAAGGAGGAGATTTTTCTCCTCCTTC）在溶液中采用单分子发夹结构(h)，具有低聚嘌呤-低聚嘧啶（Pu-Py）体系。当h与d(CTTCCTCCTCT) （s1）混合时，两条链在pH为5的聚丙烯酰胺凝胶电泳中共迁移。如果用d(TCTCCTCCTTC) （s2）代替s1，则没有观察到这种行为，两条链分别迁移。这支持了h和s1 （h:s1）而不是h和s2形成三链结构的建议，并证实了第三条嘧啶链的链极性要求，这是这种结构所必需的。电泳迁移率数据（Ferguson图）和DNase i酶分析支持h:s1形成三螺旋，圆二色性测量表明，在三螺旋形成时，有两个负椭圆：一个较弱（. delta和epsilon）。= 80 M-1 cm-1)和一个更强的(. delta…= 210 M-1 cm-1)在212 nm。后者也在三链多核苷酸中被观察到，并且可以被认为是Py:Pu:Py DNA三重体的商标。270 nm处紫外吸收和温度测量的比较表明，三链结构的熔体具有双相轮廓。较低温度的转变是双分子的，是由于三联体的击穿产生h和s1，而较高温度的转变是单分子的，是由于发夹结构向线圈结构的转变。双相到三相的转变是合作的，完全可逆的，超变色约为10%。用三态模型分析熔化曲线，可以估计三螺旋形成的热力学参数。我们发现h:s1的双相到三相转变伴随着平均焓变化（减去质子化贡献）-73（+-）。5) kcal/mol，对应于-6.6（+-）。0.4) kcal/mol结合嘧啶，可归因于堆叠和氢键相互作用。
The 26mer oligodeoxynucleotide d(GAAGGAGGAGATTTTTCTCCTCCTTC) adopts in solution a unimolecular hairpin structure (h), with an oligopurine-oligopyrimidine (Pu-Py) stem. When h is mixed with d(CTTCCTCCTCT) (s1) the two strands co-migrate in polyacrylamide gel electrophoresis at pH 5. If s1 is substituted with d(TCTCCTCCTTC) (s2), such behavior is not observed and the two strands migrate separately. This supports the suggestion of the formation of a triple-stranded structure by h and s1 (h:s1) but not by h and s2, and confirms the strand polarity requirement of the third pyrimidine strand, which is necessary for this type of structure. The formation of a triple helix by h:s1 is supported by electrophoretic mobility data (Ferguson plot) and by enzymatic assay with DNase I. circular dichroism measurements show that, upon triple helix formation, there are two negative ellipticities: a weaker one (.DELTA..epsilon. = 80 M-1 cm-1) at 242 nm and a stronger one (.DELTA..epsilon. = 210 M-1 cm-1) at 212 nm. The latter has been observed also in triple-stranded polynucleotides, and can be considered as the trademark for a Py:Pu:Py DNA triplex. Comparison of ultraviolet absorption at 270 nm and temperature measurements shows that the triple-stranded structure melts with a biphasic profile. The lower temperature transition is bimolecular and is attributable to the breakdown of the triplex to give h and s1, while the higher temperature transition is monomolecular and is due to the transition of hairpin to coil structure. The duplex-to-triplex transition is co-operative, fully reversible and with a hyperchromism of about 10%. The analysis of the melting curves, with a three-state model, allows estimation of the thermodynamic parameters of triple helix formation. We found that the duplex-to-triplex transition of h:s1 is accompanied by an average change in enthalpy (less the protonation contribution) of -73(.+-. 5) kcal/mol of triplex, which corresponds to -6.6(.+-. 0.4) kcal/mol of binding pyrimidine, attributable to stacking and hydrogen bonding interactions.