Sensing the DNA-mismatch tolerance of catalytically inactive Cas9 via barcoded DNA nanostructures in solid-state nanopores.

Autor: Sandler SE; Cavendish Laboratory, University of Cambridge, Cambridge, UK., Weckman NE; 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., Yorke S; Cavendish Laboratory, University of Cambridge, Cambridge, UK.; Yusuf Hamied Department of Chemistry, Cambridge, UK., Das A; Department of Pathology, University of Cambridge, Cambridge, UK.; Department of Chemical Engineering, Imperial College London, London, UK., Chen K; Cavendish Laboratory, University of Cambridge, Cambridge, UK., Gutierrez R; Oxford Nanopore Technologies, Oxford Science Park, Oxford, UK., Keyser UF; Cavendish Laboratory, University of Cambridge, Cambridge, UK. ufk20@cam.ac.uk.
Jazyk: angličtina
Zdroj: Nature biomedical engineering [Nat Biomed Eng] 2024 Mar; Vol. 8 (3), pp. 325-334. Date of Electronic Publication: 2023 Aug 07.
DOI: 10.1038/s41551-023-01078-2
Abstrakt: 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.
(© 2023. The Author(s).)
Databáze: MEDLINE
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