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. 2018 Sep 24;57(39):12896-12900.
doi: 10.1002/anie.201807593. Epub 2018 Sep 3.

Fluorescence Probes for ALKBH2 Allow the Measurement of DNA Alkylation Repair and Drug Resistance Responses

David L Wilson  1 , Andrew A Beharry  2 , Avinash Srivastava  3 , Timothy R O'Connor  3 , Eric T Kool  1
Affiliations

Fluorescence Probes for ALKBH2 Allow the Measurement of DNA Alkylation Repair and Drug Resistance Responses

David L Wilson et al. Angew Chem Int Ed Engl. .

Abstract

The DNA repair enzyme ALKBH2 is implicated in both tumorigenesis as well as resistance to chemotherapy in certain cancers. It is currently under study as a potential diagnostic marker and has been proposed as a therapeutic target. To date, however, there exist no direct methods for measuring the repair activity of ALKBH2 in vitro or in biological samples. Herein, we report a highly specific, fluorogenic probe design based on an oligonucleotide scaffold that reports directly on ALKBH2 activity both in vitro and in cell lysates. Importantly, the probe enables the monitoring of cellular regulation of ALKBH2 activity in response to treatment with the chemotherapy drug temozolomide through a simple fluorescence assay, which has only previously been observed through indirect means such as qPCR and western blots. Furthermore, the probe provides a viable high-throughput assay for drug discovery.

Keywords: DNA repair; biosensors; cancer; fluorescent probes; nucleic acids.

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Conflict of interest statement

Conflict of interest

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The 1-methyladenosine quenching (MAQ) probe design used to detect demethylation by ALKBH2. The pyrene excimer is quenched by photo-induced charge transfer to the damaged base. Following repair of the positively charged lesion, the quenching is lost, resulting in fluorescence emission.
Figure 2
Figure 2
Fold change in fluorescence intensity of a) probes 1–4 or b) probes 9 and 12 (1 µm) incubated with ALKBH2 (0.5 µm) measured at 385 nm (a) or 480 nm (b). c) Selectivity of probe 12 with buffer alone or supplemented with 1.5 mm MgCl2. See Supporting Information for full reaction conditions.
Figure 3
Figure 3
Probe 13 used in high-throughput format for evaluating an inhibitor. Dose-response curve of broad-spectrum demethylase inhibitor pyridine 2,4-dicarboxylic acid (PDCA) with ALKBH2 generated using probe 13. The curve yielded an IC50 measurement of 2.43 ± 0.12 µm.
Figure 4
Figure 4
Nuclease-protected probe 13p yields ALKBH2-specific signals in mammaliancell lysates. Shown are fluorescence responses of probe 13p (2 μm)with MEF lysates (1 mgmL−1 total protein). Data collected from 384-well plates over 27 hincubated at 378°C.
Figure 5
Figure 5
Nuclease-protected probe 13p reports on ALKBH2 activity in lysates derived from human cell lines. Experiments were performed in a384-well plate with 1mgmL−1 total cellular protein, 100 μm TMZ, and fluorescence was measured after 24 h incubation.
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