Advancing Topoisomerase Research Using DNA Nanotechnology

Abstract

In this Perspective, the use of DNA nanotechnology is explored as a powerful tool for studying a family of enzymes known as topoisomerases. These enzymes regulate DNA topology within a living cell and play a major role in the pharmaceutical field, serving as anti-cancer and anti-bacterial targets. This Perspective will provide a short historical overview of the methods employed in studying these enzymes and emphasizing recent advancements in assays using DNA nanotechnology. These innovations have substantially improved accuracy and expanded the understanding of enzyme activity. This perspective will showcase the versatile utility of DNA nanotechnology in advancing scientific knowledge and its application in exploring new drug candidates, particularly in the study of topoisomerase enzymes.

Keywords: DNA nanotechnology; DNA topology; drug discovery; enzymatic assays; high‐thruput screening; topoisomerases.

Figure 1

Plasmid‐based assays–Illustrations of a) Gel electrophoresis: Relaxed and supercoiled DNA molecules have different mobilities within a gel matrix. Both the supercoiled and the relaxed DNA topology has several possible configurations, resulting in the formation of a topoisomer ladder. b) Plasmid degradation: T5 exonuclease degrades only supercoiled plasmid while relaxed plasmid remains intact. The difference can be visualized using non‐specific intercalating dyes such as EthD‐. Figure created based on the concepts presented in ref. [46]. c) Fluorophore‐Quencher pair on a plasmid, the distance between the fluorophore and quencher in the (AT)n sequence alters when plasmids transition between supercoiled and relaxed states. The assay from ref. [40] presents higher fluorescence when the plasmids were supercoiled (left), while ref. [43] presents higher fluorescence when the plasmids were relaxed (right), due to different reaction conditions. Left‐ Reproduced with permission,^{[ 40 ]} copyright 2013, Oxford Academic. Right–Reproduced with permission,^{[ 43 ]} Copyright 2016, Springer Nature. d) Intercalating dye assay: Variations in DNA topology influence the binding affinity of intercalating dyes. Figure created based on the concepts presented in ref. [35]. e) Triplex formation: Plasmids form intermolecular triplexes with supercoiled or relaxed DNA. According to ref. [38] triplex DNA forms when the plasmid is supercoiled (down) and is captured on a microtiter plate surface using an oligonucleotide tethered to the surface.^{[ 38 ]} The signal is then detected using intercalating dyes or by measuring radiolabeled oligonucleotides. Ref. [39] presents the preferred triplex formation on the relaxed form of a plasmid, with the third oligonucleotide labeled with a fluorescence tag (up). The difference in triplex formation efficiency results from different reaction conditions. Up–Reproduced with permission,^{[ 39 ]} Copyright 2010, Elsevier. Down‐ figure created based on the concepts presented in ref. [38].

Figure 2

Synthetic DNA nanoprobes–Illustrations of a) Single‐stranded Trefoil knot construction, which serves as the substrate for bacterial topo I and III. Figure created based on the concepts presented in ref. [48]. b) Small DNA probes on a 2D DNA origami surface to study topoisomerase IB secondary binding. Reproduced with permission,^{[ 53 ]} copyright 2010, ACS nano. c) Incorporation of quantum dots with nanoprobes takes advantage of the distinct photophysical characteristics of quantum dots to produce visible fluorescence recovery when specifically cleaved by mycobacterial topoisomerase I. Reproduced with permission,^{[ 51 ]} copyright 2016, Royal Society of Chemistry. d) Hairpin‐shaped nano DNA sensor with a quencher‐fluorophore pair. After reaction with hTopI at a specific cleavage site, the quencher and fluorophore separate, allowing fluorescence detection. Reproduced with permission,^{[ 50 ]} copyright 2013, MDPI.

Figure 3

Amplification‐based assays – Illustrations of a) HTS for new drug candidates. b) dumbbell‐shaped nanoprobe for hTopI detection, followed by RCA reaction upon the surface. Reproduced with permission,^{[ 55 ]} copyright 2009, American Chemical Society. c) single‐stranded trefoil knot as a substrate for bacterial topoisomerase IA, followed by RCA reaction within a solution. Figure created based on the concepts presented in ref. [62].