Multiplexed Detection of Site Specific Recombinase and DNA Topoisomerase Activities at the Single Molecule Level

Abstract

We previously demonstrated 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 was achieved by topoisomerase I mediated closure of a nicked DNA circle followed by rolling circle amplification leading to an anchored product that was visualized at the single molecule level by hybridization to fluorescently labeled probes (Stougaard et al. ACS Nano 2009, 3, 223-33). An important inherent property of the presented setup is, at least in theory, the easy adaptability to multiplexed enzyme detection simply by using differently labeled probes for the detection of rolling circle products of different circularized substrates. In the present study we demonstrate the specific detection of three different enzyme activities, human topoisomerase I, and Flp and Cre recombinase in nuclear extracts from human cells one at a time or multiplexed using the rolling circle amplification based single-molecule detection system. Besides serving as a proof-of-principle for the feasibility of the presented assay for multiplexed enzyme detection in crude human cell extracts, the simultaneous detection of Flp and Cre activities in a single sample may find immediate practical use since these enzymes are often used in combination to control mammalian gene expression.

Extraction of active enzymes from “hard-to-break-cells”: Evaluation by a RCA-based assay

“…Each RCP is optically detected at the single molecule level by hybridization to fluorescently labeled detection probes followed by microscopic analysis, shown in Figure 1b. Compared to other single-molecule activity assays for TopI, in which magnetic tweezers, optical trapping, or other specialized setups are used to manipulate DNA, the RCA-based system is amenable for convenient enzyme activity screening as well as for multiplexed detection, using fluorescence probes with differentiable emissions to detect RCPs generated by different DNA substrates specific to different enzyme activities [8].…”

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.…”

Microfluidics-mediated isothermal detection of enzyme activity at the single molecule level

“…The DNA substrates comprise a single oligonucleotide, which is converted to a closed DNA circle by a single enzyme cleavage-ligation event. For each circularized substrate subsequent RCA results in the creation of one rolling circle product (RCP) consisting of multiple (up to 10 3 ) tandem copies of the circularized substrate [3], [20], [21]. Each RCP is optically detected at the single molecule level by hybridization to fluorescently labeled probes followed by microscopic analysis.…”

“…The droplets were left to dry for 12 hours. Wash, RCA, and hybridization of probes were performed similarly as bulk experiments described in [3]. Microscopy Epifluorescent and bright field images were captured with an inverted fluorescence microscope (Axio Observer, Zeiss).…”

“…We recently developed a multiplexed, single molecule level, Rolling circle Enhanced Enzyme Activity Detection (REEAD) system for the scientifically and clinically important endogenous human enzyme, topoisomerase I (TopI) and the related enzymes, Flp and Cre recombinases [2], [3]. TopI exerts important cellular functions by introducing transient single-stranded DNA breaks in the human genome allowing the relaxation of superhelical tension arising as a consequence of DNA tracking processes [4].…”