Sharma Nischal, Whinn Kelsey S, Ghodke Harshad, van Oijen Antoine M, Lewis Jacob S, Spenkelink Lisanne M
Molecular Horizons and School of Chemistry and Molecular Bioscience, University of Wollongong, Wollongong, NSW, 2522, Australia.
Molecular Horizons and School of Chemistry and Molecular Bioscience, University of Wollongong, Wollongong, NSW, 2522, Australia.
Anal Biochem. 2025 Aug;703:115883. doi: 10.1016/j.ab.2025.115883. Epub 2025 Apr 25.
Rolling-circle DNA replication is a DNA-duplication mechanism whereby circular DNA templates are continuously copied to produce long DNA products. It is widely used in molecular diagnostics, DNA sequencing, nanotechnology, and in vitro DNA replication studies. The efficiency of rolling-circle replication reaction heavily relies on the quality of the rolling-circle DNA template. Existing methods to create rolling-circle DNA substrates often rely on unique restriction sites and have limited control over replication fork topology and position. To address these limitations, we present a straightforward, customizable, and efficient strategy for producing rolling-circle DNA substrates with control over gap size and fork position. Our method relies on the use of nickase Cas9 (nCas9), which can be programmed to target specific DNA sequences using guide RNAs. In a one-pot reaction, we target nCas9 to four sites on an 18-kb plasmid to create 8-11-bp fragments. These fragments are removed and a flap oligo is ligated, to construct a fork with precisely controlled flap length and gap size. We demonstrate the application of this DNA substrate in an in vitro single-molecule rolling-circle DNA-replication assay. With our method, any plasmid DNA can be converted into a rolling-circle template, permitting generation of more physiologically-relevant DNA templates.
滚环DNA复制是一种DNA复制机制，通过该机制，环状DNA模板被连续复制以产生长链DNA产物。它广泛应用于分子诊断、DNA测序、纳米技术和体外DNA复制研究。滚环复制反应的效率在很大程度上依赖于滚环DNA模板的质量。现有的创建滚环DNA底物的方法通常依赖于独特的限制性酶切位点，并且对复制叉拓扑结构和位置的控制有限。为了解决这些限制，我们提出了一种直接、可定制且高效的策略，用于生产能够控制缺口大小和叉位置的滚环DNA底物。我们的方法依赖于切口酶Cas9（nCas9）的使用，它可以通过引导RNA编程靶向特定的DNA序列。在一锅反应中，我们将nCas9靶向一个18 kb质粒上的四个位点，以产生8-11 bp的片段。这些片段被去除，然后连接一个瓣状寡核苷酸，以构建一个具有精确控制的瓣长度和缺口大小的叉。我们展示了这种DNA底物在体外单分子滚环DNA复制测定中的应用。通过我们的方法，任何质粒DNA都可以转化为滚环模板，从而允许生成更多与生理相关的DNA模板。
