Recording of DNA Knots Formation and Their Splitting by TOPII with Solid-state Nanopore Devices

Abstract

DNA knots represent critical topological structures arising during DNA replication, recombination, and repair, of which the aberrant accumulation threatens genomic stability. Helicases and topoisomerases act cooperatively to maintain genomic integrity by specifically recognizing DNA knots and progressively unwinding them or relieving topological stress through transient DNA strand cleavage. This study employs solid-state nanopore (SSN) technology to achieve high-resolution detection of λ-DNA knots under optimized conditions. Our work demonstrates that DNA knot translocation generates multistage signals of ionic current blockade, with blockage current amplitude positively correlating with topological complexity. Furthermore, the capture frequency of DNA knots increases significantly with applied bias voltages, while translocation duration decreases with reduced channel length (referring to the thickness of silicon nitride). Notably, topoisomerase II (TOPII) exhibits moderate resolving activity toward DNA knots, manifested by an increased proportion of linear DNA signals and shortened blockade durations. The splitting efficiency of TOPII is influenced by the supercoiling status of DNA and the concentration of enzyme; the underlying molecular mechanism needs further investigation with combined single-molecule fluorescence techniques. This study innovatively applies solid-state nanopore technology to explore DNA topology and DNA-TOPII interaction, providing a new research strategy for related fields.

Keywords

DNA knot / Single-molecule conformation / Topoisomerase II (TOPII) / DNA-protein interaction / Solid-state nanopore

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Jilin University, The Editorial Department of Chemical Research in Chinese Universities and Springer-Verlag GmbH

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