Department of Chemical and Biological Engineering, Chalmers University of Technology, S41296 Goteborg, Sweden.
Comput Struct Biotechnol J. 2014 Sep 3;11(18):66-72. doi: 10.1016/j.csbj.2014.08.011. eCollection 2014 Aug.
Studies of DNA-ligand interaction on a single molecule level provide opportunities to understand individual behavior of molecules. Construction of DNA molecules with repetitive copies of the same segments of sequences linked in series could be helpful for enhancing the interaction possibility for sequence-specific binding ligand to DNA. Here we report on the use of synthetic oligonucleotides to self-assembly into duplex DNA concatemeric molecules. Two strands of synthetic oligonucleotides used here were designed with 50-mer in length and the sequences are semi-complimentary so to hybridize spontaneously into concatemers of double stranded DNA. In order to optimize the length of the concatemers the oligonucleotides were incubated at different oligomer concentrations, ionic strengths and temperatures for different durations. Increasing the salt concentration to 200 mM NaCl was found to be the major optimizing factor because at this enhanced ionic strength the concatemers formed most quickly and the other parameters had no detectable effect. The size and shape of formed DNA concatemers were studied by gel electrophoresis in agarose, polyacrylamide gels and by AFM. Our results show that linear DNA constructs up to several hundred base pairs were formed and could be separated from a substantial fraction of non-linear constructs.
在单分子水平上研究 DNA-配体相互作用为理解分子的个体行为提供了机会。构建具有串联连接的相同序列重复片段的 DNA 分子可以有助于增强序列特异性结合配体与 DNA 的相互作用可能性。在这里，我们报告了使用合成寡核苷酸自组装成双链 DNA 串联分子。这里使用的两条合成寡核苷酸设计为 50 个碱基长，序列是半互补的，因此可以自发杂交成双链 DNA 的串联物。为了优化串联物的长度，将寡核苷酸在不同的寡核苷酸浓度、离子强度和温度下孵育不同的时间。发现增加盐浓度至 200 mM NaCl 是主要的优化因素，因为在这种增强的离子强度下，串联物形成得最快，而其他参数没有可检测到的影响。通过琼脂糖、聚丙烯酰胺凝胶电泳和 AFM 研究了形成的 DNA 串联物的大小和形状。我们的结果表明，形成了长达数百个碱基对的线性 DNA 结构，并且可以与大部分非线性结构分离。
Zotero 插件
