Liang Xingguo, Kuhn Heiko, Frank-Kamenetskii Maxim D
Center for Advanced Biotechnology and Department of Biomedical Engineering, Boston University, Massachusetts 02215, USA.
Biophys J. 2006 Apr 15;90(8):2877-89. doi: 10.1529/biophysj.105.074104. Epub 2006 Feb 3.
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.
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。为了阐明其功能，必须更好地了解ssDNA的结构。然而，由于ssDNA极其多变的结构特征，现有的数据过于有限，无法清晰地呈现ssDNA的全貌。在本研究中，通过形成DNA连环体并通过电泳分析确定其拓扑结构（连接数，Lk），我们证明了对由两个ssDNA分子形成的连环体的研究可以产生有关ssDNA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，由两个短的（60/70 nt）ssDNA分子构建连环体。两个DNA环之间形成的二级结构决定了构建的DNA连环体拓扑结构（Lk值）。因此，通过观察连接数随序列和条件的变化，实验监测二级结构的形成。我们发现，ssDNA的二级结构比预期更容易形成：折叠的ssDNA结构内部环中的两条链更倾向于相互缠绕，而不是分开形成一个环，并且仅包含错配碱基对的双链体可以在生理条件下形成。
