Fluorescence-free quantification of protein/nucleic-acid binding through single-molecule kinetic locking
Fluorescence-free quantification of protein/nucleic-acid binding through single-molecule kinetic locking
ABSTRACT 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 new 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 E. coli RecQ helicase with its DNA substrate.
Ducos Bertrand、Allemand Jean-Fran?ois、Rieu Martin、Croquette Vincent、Valle-Orero Jessica
Laboratoire de physique de L?ˉ¨|cole Normale Sup¨|rieure de Paris, CNRS, ENS, Universit¨| PSL, Sorbonne Universit¨|, Universit¨| de ParisLaboratoire de physique de L?ˉ¨|cole Normale Sup¨|rieure de Paris, CNRS, ENS, Universit¨| PSL, Sorbonne Universit¨|, Universit¨| de ParisLaboratoire de physique de L?ˉ¨|cole Normale Sup¨|rieure de Paris, CNRS, ENS, Universit¨| PSL, Sorbonne Universit¨|, Universit¨| de ParisLaboratoire de physique de L?ˉ¨|cole Normale Sup¨|rieure de Paris, CNRS, ENS, Universit¨| PSL, Sorbonne Universit¨|, Universit¨| de ParisLaboratoire de physique de L?ˉ¨|cole Normale Sup¨|rieure de Paris, CNRS, ENS, Universit¨| PSL, Sorbonne Universit¨|, Universit¨| de Paris
生物科学研究方法、生物科学研究技术生物物理学分子生物学
Ducos Bertrand,Allemand Jean-Fran?ois,Rieu Martin,Croquette Vincent,Valle-Orero Jessica.Fluorescence-free quantification of protein/nucleic-acid binding through single-molecule kinetic locking[EB/OL].(2025-03-28)[2025-05-18].https://www.biorxiv.org/content/10.1101/2020.09.30.321232.点此复制
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