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Single-molecule kinetic locking allows fluorescence-free quantification of protein/nucleic-acid binding.

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 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.

SUBMITTER: Rieu M

PROVIDER: S-EPMC8443601 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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Single-molecule kinetic locking allows fluorescence-free quantification of protein/nucleic-acid binding.

Rieu Martin M Valle-Orero Jessica J Ducos Bertrand B Allemand Jean-François JF Croquette Vincent V

Communications biology 20210915 1

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 validat ...[more]

PMID: 34526657

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