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bioRxiv · 10.1101/2025.05.30.656938

Nanopore Assay for Fingerprinting DNA Binding and Quantifying Real-Time Cleavage by Catalytically Active Cas9 Enzyme

Nagpure, P.·Suresh, S.·Shet, D.·Soni, G. V.
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Abstract

Nanopore sensing, a high-resolution DNA sequencing technology, is fast expanding into novel and exciting direction of probing specific DNA-enzyme interactions. Although proven excellent for detection of structural features of bare DNA, quantitative measurements on enzyme-DNA complexes and its 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 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. TOC Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/656938v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@9ea40org.highwire.dtl.DTLVardef@f214e6org.highwire.dtl.DTLVardef@13a10bborg.highwire.dtl.DTLVardef@12067ab_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Nagpure, P., Suresh, S., Shet, D., Soni, G. V.. 2025-05-30. Nanopore Assay for Fingerprinting DNA Binding and Quantifying Real-Time Cleavage by Catalytically Active Cas9 Enzyme. https://doi.org/10.1101/2025.05.30.656938

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