Laboratoire de physique de l'Ecole Normale Supérieure (LPENS), ENS, Université PSL, CNRS, Sorbonne Université, Université de Paris, Paris, France.
Institut de Biologie de l'Ecole Normale Supérieure (IBENS), ENS, Université PSL, CNRS, INSERM, Paris, France.
Commun Biol. 2021 Sep 15;4(1):1083. doi: 10.1038/s42003-021-02606-z.
Fluorescence-free micro-manipulation of nucleic acids (NA) allows the functional characterization of DNA/RNA processing proteins, without the interference of labels, but currently fails to detect and quantify their binding. To overcome this limitation, we developed a method based on single-molecule force spectroscopy, called kinetic locking, that allows a direct in vitro visualization of protein binding while avoiding any kind of chemical disturbance of the protein's natural function. We validate kinetic locking by measuring accurately the hybridization energy of ultrashort nucleotides (5, 6, 7 bases) and use it to measure the dynamical interactions of Escherichia coli/E. coli RecQ helicase with its DNA substrate.
无荧光标记的核酸（NA）微操控技术可在不干扰标记的情况下对 DNA/RNA 加工蛋白进行功能表征，但目前还无法检测和定量其结合。为了克服这一限制，我们开发了一种基于单分子力谱学的方法，称为动力学锁定，该方法可在避免任何化学干扰蛋白质天然功能的情况下，直接体外可视化蛋白质结合。我们通过准确测量超短核苷酸（5、6、7 个碱基）的杂交能来验证动力学锁定，并将其用于测量大肠杆菌/大肠杆菌 RecQ 解旋酶与其 DNA 底物的动态相互作用。
