Cavendish Laboratory, University of Cambridge, Cambridge, UK.
Institute for Studies in Transdisciplinary Engineering Education & Practice, Department of Chemical Engineering & Applied Chemistry, University of Toronto, Toronto, Canada.
Nat Biomed Eng. 2024 Mar;8(3):325-334. doi: 10.1038/s41551-023-01078-2. Epub 2023 Aug 7.
Single-molecule quantification of the strength and sequence specificity of interactions between proteins and nucleic acids would facilitate the probing of protein-DNA binding. Here we show that binding events between the catalytically inactive Cas9 ribonucleoprotein and any pre-defined short sequence of double-stranded DNA can be identified by sensing changes in ionic current as suitably designed barcoded linear DNA nanostructures with Cas9-binding double-stranded DNA overhangs translocate through solid-state nanopores. We designed barcoded DNA nanostructures to study the relationships between DNA sequence and the DNA-binding specificity, DNA-binding efficiency and DNA-mismatch tolerance of Cas9 at the single-nucleotide level. Nanopore-based sensing of DNA-barcoded nanostructures may help to improve the design of efficient and specific ribonucleoproteins for biomedical applications, and could be developed into sensitive protein-sensing assays.
单分子定量分析蛋白质与核酸之间相互作用的强度和序列特异性，将有助于探测蛋白-DNA 结合。本文中，我们展示了通过检测离子电流的变化，可以识别出具有 Cas9 结合双链 DNA 突出端的经适当设计的带条形码线性 DNA 纳米结构与无活性 Cas9 核糖核蛋白之间的结合事件，这些纳米结构可以通过固态纳米孔进行转位。我们设计了带条形码的 DNA 纳米结构，以在单核苷酸水平上研究 DNA 序列与 Cas9 的 DNA 结合特异性、DNA 结合效率和 DNA 错配容忍度之间的关系。基于纳米孔的 DNA 条形码纳米结构检测，可能有助于改进用于生物医学应用的高效且特异性的核糖核蛋白的设计，并可进一步开发为灵敏的蛋白质检测分析方法。
