Monitoring single-stranded DNA secondary structure formation by determining the topological state of DNA catenanes
Monitoring single-stranded DNA secondary structure formation by determining the topological state of DNA catenanes
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DOI：
10.1529/biophysj.105.074104
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发表时间：
2006-04-01
影响因子：
3.4
通讯作者：
Frank-Kamenetskii, MD
中科院分区：
文献类型：
--
作者：
Liang, XG;Kuhn, H;Frank-Kamenetskii, MD
单链 DNA (ssDNA) 在 DNA 复制、重组、修复和转录过程中具有重要的生物学功能。必须更好地理解 ssDNA 的结构才能阐明其功能。然而，由于单链DNA极其反复无常的结构特征，现有数据过于有限，无法清晰地描述单链DNA。在本研究中，通过形成DNA链烷并通过电泳分析确定其拓扑结构（连接数，Lk），我们证明对两个单链DNA分子形成的链烷的研究可以产生有关单链DNA二级结构的有价值的新信息。我们通过线性寡脱氧核苷酸的酶促环化，用两个短 (60/70 nt) ssDNA 分子构建索烷。两个DNA环之间形成的二级结构决定了所构建的DNA链烷的拓扑结构（Lk值）。因此，通过观察连接数随序列和条件的变化来实验监测二级结构的形成。我们发现单链DNA的二级结构比预期更容易形成：折叠单链DNA结构中内部环中的两条链更喜欢相互编织而不是单独形成环，并且在生理条件下可以形成仅包含错配碱基对的双链体。
Single-stranded DNA (ssDNA) has essential biological functions during DNA replication, recombination, repair, and transcription. The structure of ssDNA must be better understood to elucidate its functions. However, the available data are too limited to give a clear picture of ssDNA due to the extremely capricious structural features of ssDNA. In this study, by forming DNA catenanes and determining their topology (the linking number, Lk) through the electrophoretic analysis, we demonstrate that the studies of catenanes formed from two ssDNA molecules can yield valuable new information about the ssDNA secondary structure. We construct catenanes out of two short (60/70 nt) ssDNA molecules by enzymatic cyclization of linear oligodeoxynucleotides. The secondary structure formed between the two DNA circles determines the topology (the Lk value) of the constructed DNA catenane. Thus, formation of the secondary structure is experimentally monitored by observing the changes of linking number with sequences and conditions. We found that the secondary structure of ssDNA is much easier to form than expected: the two strands in an internal loop in the folded ssDNA structure prefer to braid around each other rather than stay separately forming a loop, and a duplex containing only mismatched basepairs can form under physiological conditions.