The kinetics of oligonucleotide replacements
The kinetics of oligonucleotide replacements
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DOI：
10.1006/jmbi.2000.3573
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发表时间：
2000-03-24
影响因子：
5.6
通讯作者：
Lyamichev, VI
中科院分区：
文献类型：
--
作者：
Reynaldo, LP;Vologodskii, AV;Lyamichev, VI
如果两条核酸链中的一条形成分子内或分子间二级结构，则两条核酸链之间的双链体的形成受到限制。新双链体的形成需要初始结构的解离和置换。为了理解这种类型的动力学的机制，我们研究了用相同序列的未标记的探针替换与互补DNA靶结合的标记的DNA寡核苷酸探针。使用凝胶位移测定法测量12、14和16-核苷酸探针的置换动力学，作为未标记探针的温度和浓度的函数。结果表明，总的置换率是两个动力学途径的组合：解离和顺序置换。解离途径通过初始双链体的自发解离发生，随后是靶标和未标记探针的缔合。顺序置换途径仅需要初始双链体的部分解链以允许与未标记探针形成分支成核复合物，随后通过分支点的迁移完全置换标记探针。来自解离途径的贡献在接近标记探针的熔点的温度下占主导地位，而来自置换途径的贡献在较低温度下和当置换的未标记探针的浓度高时占主导地位。结果表明，在生理条件下，单链寡核苷酸探针和靶分子的结构化区域之间的双链体形成主要通过顺序置换机制发生。(C)北京大学出版社.
The formation of a duplex between two nucleic acid strands is restricted if one of the strands forms an intra- or intermolecular secondary structure. The formation of the new duplex requires the dissociation and replacement of the initial structure. To understand the mechanism of this type of kinetics we studied the replacement of a labeled DNA oligonucleotide probe bound to a complementary DNA target with an unlabeled probe of the same sequence. The replacement kinetics were measured using a gel-shift assay for 12, 14 and 16-nucleotide probes as a function of temperature and concentration of the unlabeled probe. The results demonstrate that the overall replacement rate is a combination of two kinetic pathways: dissociative and sequential displacement. The dissociative pathway occurs by the spontaneous dissociation of the initial duplex followed by association of the target and unlabeled probe. The sequential displacement pathway requires only the partial melting of the initial duplex to allow for the formation of a branched nucleation complex with the unlabeled probe, followed by the complete displacement of the labeled probe by migration of the branch point. The contribution from the dissociative pathway is predominant at temperatures close to the melting point of the labeled probe, whereas the contribution from the displacement pathway prevails at lower temperatures and when the concentration of the replacing unlabeled probe is high. The results show that at physiological conditions, duplex formation between a single-stranded oligonucleotide probe and a structured region of a target molecule occurs mainly by the sequential-displacement mechanism. (C) 2000 Academic Press.