Single-Molecule Detection of Human Topoisomerase I Cleavage−Ligation Activity

Abstract

In the present study, we demonstrate the conversion of a single human topoisomerase I mediated DNA cleavage-ligation event happening within nanometer dimensions to a micrometer-sized DNA molecule, readily detectable using standard fluorescence microscopy. This conversion is achieved by topoisomerase I mediated closure of a nicked DNA dumbbell structure, followed by rolling circle amplification. The resulting product consists of multiple tandem repeats of the DNA dumbbell and can subsequently be visualized by annealing to fluorescently labeled probes. Since amplification involves no thermal cycling, each fluorescent rolling circle product, which gives rise to an individual signal upon microscopic analysis, will correspond to a single human topoisomerase I mediated cleavage-ligation event. Regarding sensitivity, speed, and ease of performance, the presented activity assay based on single-molecule product detection is superior to current state of the art assays using supercoiled plasmids or radiolabeled oligonucleotides as the substrate for topoisomerase I activity. Moreover, inherent in the experimental design is the easy adaptation to multiplexed and/or high-throughput systems. Human topoisomerase I is the cellular target of clinically important anticancer drugs, and the effect of such drugs corresponds directly to the intracellular topoisomerase I cleavage-ligation activity level. We therefore believe that the presented setup, measuring directly the number of cleavage-ligation events in a given sample, has great diagnostic potential, adding considerably to the possibilities of accurate prognosis before treatment with topoisomerase I directed chemotherapeutics.

Correlation between topoisomerase I and tyrosyl-DNA phosphodiesterase 1 activities in non-small cell lung cancer tissue

“…1999; Husain et al, 1994). Since a comparison of the TOP1 activity in the tumor and adjacent non-tumor tissue has not been performed in NSCLC we measured TOP1 activity in the cryosection extracts also used for TDP1 measurement using our previously published TOP1 nanosensor (Stougaard et al, 2009). We found that there was a significantly (p = 0.0038) higher activity of TOP1 in the tumor samples compared to the non-tumor samples (Fig.…”

“…Using our previously developed nanosensors capable of measuring the specific activities of TOP1 (Stougaard et al, 2009) and TDP1 (Jensen et al, 2013) we examined the activities of TOP1 and TDP1 in cryosections from 24 paired (tumor and adjacent non-tumor) NSCLC tissues. We found that both TOP1 and TDP1 were upregulated in the tumor tissue compared to the adjacent non-tumor tissue in NSCLC.…”

“…The TOP1 activity assay was carried out essentially as described in Stougaard et al (Stougaard et al, 2009). For testing the influence of Tissue-Tek on the assay, the reaction was performed in 1 × TDP1-buffer supplemented with 1 μL purified TOP1, 0.2 μM TOP1-Id16, and different concentrations of Tissue-Tek (0%, 0.625%, 1.25%, 2.5%, 5%, and 10%).…”

DNA-based nanosensors for next-generation clinical diagnostics via detection of enzyme activity

“…Detection of enzyme activities via DNA nanosensors usually takes advantages of the functionality of DNA-modifying enzyme, such as DNA cleavage, ligation, synthesis and repair [15]. For example, specific and sensitive measurements of the cleavage-religation activities from the DNA topoisomerase and the tyrosine recombinase families have previously been demonstrated, by combining DNA nanosensors with rolling circle amplification [16,17]. Even though this assay enables a single molecular sensitivity, the highly delicate fluorescence microscope and the time-consuming procedures have limited its applicability for point-of-care settings.…”

Real-time detection of TDP1 activity using a fluorophore–quencher coupled DNA-biosensor

“…Hence, besides the obvious interest to basic science there is a growing interest for biosensors capable of detecting the specific action of enzymes in complex biological samples such as crude cell extracts used for clinical applications. In line with this demand we have previously developed DNA-based biosensors capable of detecting the specific activity of human topoisomerase I (TopI), and the two related enzymes, Flp-and Cre recombinases, in crude cell extract Stougaard et al, 2009). These biosensors relied on rolling circle amplification (RCA) and allowed highly sensitive and quantitative detection of enzyme activities at the level of single catalytic events even in single cells .…”