基于DNA折纸的DNA复制的单分子检测和表征

Single-molecule detection and characterization of DNA replication based on DNA origami

目的探索DNA复制的单分子检测和表征途径。方法单链DNA模板链的两端通过碱基互补配对固定在DNA折纸纳米结构上,利用原子力显微镜(AFM)在单个DNA分子水平上对复制过程中的DNA分子的不同阶段进行检测和表征,其中包括:复制前后的形貌,复制过程中大肠杆菌DNA聚合酶ⅠKlenow Fragment片段(KF)的分布,以及复制后Biotin-Streptavidin(BA)分子识别反应在单个DNA链上所引起的进一步形貌变化。同时,采用常规的琼脂糖凝胶电泳方法分析DNA复制前后的变化情况。结果(1)DNA模板链成功连结在三角折纸的特定位点,连接效率达到50%以上;(2)DNA复制过程中,KF结合在DNA模板链上,复制后KF从DNA链脱离;(3)复制前后DNA链高度变化明显,DNA链高度增加了约0.7 nm;(4)复制后,当加入Streptavidin时,其结合于含有Biotin标记的新合成DNA链位置处,形成BA复合物,平均高度达到约4.9 nm;(5)琼脂糖凝胶电泳结果也显示单链DNA变为双链,以及双链DNA分子上结合的BA复合物。结论通过将AFM与DNA折纸技术相结合,可实现单分子水平上的DNA复制的检测和表征,此方法将有助于研究DNA聚合酶的作用机制以及不同因素对DNA复制的影响。

Objective To investigate single-molecule detection and characterization of DNA replication. Methods Single-stranded DNA （ssDNA） as the template of DNA replication was attached to DNA origami by a hybridization reaction based on the complementary base-pairing principle. DNA replication catalyzed by E.coli DNA polymerase I Klenow Fragment （KF） was detected using atomic force microscopy （AFM）. The height variations between the ssDNA and the double-stranded DNA （dsDNA）, the distribution of KF during DNA replication and biotin-streptavidin （BA） complexes on the DNA strand after replication were detected. Agarose gel electrophoresis was employed to analyze the changes in the DNA after replication. Results The designed ssDNA could be anchored on the target positions of over 50% of the DNA origami. The KF was capable of binding to the ssDNA fixed on DNA origami and performing its catalytic activities, and was finally dissociated from the DNA after replication. The height of DNA strand increased by about 0.7 nm after replication. The addition of streptavidin also resulted in an DNA height increase to about 4.9 nm due to the formation of BA complexes on the biotinylated dsDNA. The resulting dsDNA and BA complex were subsequently confirmed by agarose gel electrophoresis. Conclusion The combination of AFM and DNA origami allows detection and characterization of DNA replication at the single molecule level, and this approach provides better insights into the mechanism of DNA polymerase and the factors affecting DNA replication.