Title:	Nanopore assay for fingerprinting DNA binding and quantifying real-time cleavage by catalytically active Cas9 enzyme
Authors:	Nagpure, Punitkumar Suresh, Sarangi Shet, Divya Soni, Gautam V
Keywords:	DNA Nanomachines DNA sequencing Nanosensors Nanopores Protein sequencing Nanofabrication Nanopatterning
Issue Date:	21-Nov-2025
Publisher:	Springer Nature
Citation:	Journal of Nanobitechnology, 2025, Vol. 23 (1), AR No. 728
Abstract:	Nanopore sensing, a high-resolution DNA sequencing technology, is rapidly expanding into novel and exciting directions of probing specific DNA-enzyme interactions. Although proven excellent for the detecting structural features of bare DNA, quantitative measurements on enzyme-DNA complexes and their real-time activity are lagging and only starting to emerge for long DNA templates. Signal-to-noise requirement and high translocation speeds make it difficult to detect protein bound on biologically relevant plasmid-length DNA. To this end we report accurate position detection of a catalytically active Cas9 bound to its single or multiple target sites on the DNA. Protein position is fingerprinted using event charge deficit (ECD) based analysis of the high signal-to-noise electrical signals as the complex translocates through a glass nanopore. Using a time-dependent assay, we quantify the kinetics of the released products upon enzymatic cleavage of the target DNA by the wild-type Cas9 nuclease. Our approach enables the nanopore-based single-molecule sensing of DNA-protein complexes, for real-time monitoring of biochemical reactions. This may help understand protein binding & localization as well as improve Cas9-based targeting in genome engineering applications.
Description:	Open Access
URI:	http://hdl.handle.net/2289/8667
Alternative Location:	https://www.biorxiv.org/content/10.1101/2025.05.30.656938v1 https://doi.org/10.1186/s12951-025-03837-6
Copyright:	2025 The Author(s)
Appears in Collections:	Research Papers (SCM)

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