A clickable psoralen to directly quantify DNA interstrand crosslinking and repair

Abstract

DNA interstrand crosslinks (ICLs) represent physical obstacles to advancing replication forks and transcription complexes. A range of ICL-inducing agents have successfully been incorporated into cancer therapeutics. While studies have adopted UVA-activated psoralens as model ICL-inducing agents for investigating ICL repair, direct detection of the lesion has often been tempered by tagging the psoralen scaffold with a relatively large reporter group that may perturb the biological activity of the parent psoralen. Here a minimally-modified psoralen probe was prepared featuring a small alkyne handle suitable for click chemistry. The psoralen probe, designated 8-propargyloxypsoralen (8-POP), can be activated by UVA in vitro to generate ICLs that are susceptible to post-labeling with an azide-tagged fluorescent reporter via a copper-catalyzed reaction. A modified alkaline comet assay demonstrated that UVA-activated 8-POP proficiently generated ICLs in cells. Cellular 8-POP-DNA lesions were amenable to click-mediated ligation to fluorescent reporters in situ, which permitted their detection and quantitation by fluorescence microscopy and flow cytometry. Small molecule DNA repair inhibitors to 8-POP-treated cells attenuated the removal of 8-POP-DNA lesions, validating 8-POP as an appropriate probe for investigating cellular ICL repair. The post-labeling strategy applied in this study is inexpensive, rapid and highly modular in nature with the potential for multiple applications in DNA repair studies.

Keywords: Chemical screening; Click chemistry; DNA repair; Imaging; Interstrand DNA crosslink; Psoralen.

Figure 1

(A) A schematic representation of the post-labeling strategy adopted in this report. In this scheme, 8-POP, an analogue of the clinically-applied 8-MOP, is activated by UVA to generate covalent 8-POP-DNA adducts. The alkyne-appended 8-POP-DNA lesion may be post-labeled via ligation with an azide-tagged fluorescent reporter (designated here as N₃-linker-FT) using a Cu(I)-catalyzed dipolar cycloaddition. The level of 8-POP-DNA lesions can readily be quantified by measuring fluorescence. (B) The synthetic route used to produce 8-POP.

Figure 2

8-POP can generate DNA ICLs following activation by UVA light in vitro. (A) Linearized pEGFPPCNA plasmid DNA was initially reacted with 0 (NT) or 31 μM 8-POP and exposed to UVA light for 0 – 30 min as indicated. Each sample was then subjected to denaturation and electrophoresis as described in the Materials and Methods. Controls that were not thermally denatured are denoted by a C and are representative of double stranded linearized plasmid DNA. (B) The fraction of double stranded DNA stabilized by 8-POP represented in (A) was quantitated and is expressed as a function of time of UVA exposure.

Figure 3

The ligation of 8-POP-modified plasmid DNA with an azide-tagged fluorescent reporter via click chemistry. Supercoiled pEGFP-PCNA plasmid DNA was initially reacted with 50 μM 8-POP and UVA for 30 min. The contents of each reaction were then subjected to conjugation with Alexa azide fluor 546 via Cu(I)-catalyzed click chemistry using 0 – 5 mM sodium ascorbate or TCEP. Samples were fractionated through agarose gels by electrophoresis and the gels scanned for Alexa fluorescence (top panel). Gels were then counterstained with SYBR green (bottom panel) and scanned to reveal the total DNA content of each sample. M denotes molecular weight markers.

Figure 4

8-POP generates DNA ICLs in HeLa cells following UVA exposure. HeLa cells were initially treated with DMSO vehicle, 50 μM 8-MOP or 8-POP as indicated for 60 min and then exposed to 0 or 100 J/m² UVA. Following irradiation, samples were subjected to treatment with 200 μM H₂O₂ or PBS as a control for 15 min at 37°C. Samples were then processed and assayed for their levels of DNA damage using an alkaline comet assay as detailed in the Materials and Methods section. Each column represents the mean level of DNA damage for each sample as reflected by the visual score in arbitrary units ± SEM. At least 100 comets were scored per sample.

Figure 5

8-POP-DNA lesions can be detected within HeLa cells in situ. Representative microscopy images of HeLa cells that were treated with 50 μM 8-POP for 60 min and then irradiated with 0 J/m² (top panels) or 100 J/m² (bottom panels). Samples were subsequently pre-extracted, fixed and subjected to post-labeling with an azide-tagged red fluorescent reporter as described in the Materials and Methods. Samples were counterstained with DAPI, mounted on glass slides and then visualized by epi-fluorescence microscopy using DAPI and TRITC filters to define the nucleus and reveal the red 8-POP adduct staining pattern, respectively.

Figure 6

The post-labeling strategy of 8-POP can be applied to measure the cellular repair of 8-POP-DNA adducts and its inhibition by small molecules. (A and B) HeLa cells were exposed to 50 μM 8-POP for 60 min and then irradiated with 100 J/m² UVA. Cells were subsequently released into media containing DMSO vehicle or 40 μM aphidicolin, olaparib or T2AA for 24 h. All samples were then hypotonically lysed, fixed, subjected to post-labeling with an azide-tagged Alexa 488 reporter by click chemistry, counterstained with propidium iodide and analyzed by flow cytometry as described in the Materials and Methods. (A) A representative histogram displaying the relative levels of 8-POP-DNA adducts as reflected by the Alexa 488 signal immediately following UVA irradiation (“start”, solid black histogram) and 24 h after release into media containing DMSO as a control (light grey histogram) or 40 μM T2AA (medium grey histogram). (B) A quantitative representation of the histogram displaying the median ICL (Alexa 488) signal as a function of treatment. (C) HT1080 cells were initially treated with 8-POP and UVA as described for (A and B) and then released into media containing DMSO vehicle or 62.5 – 125 nM gemcitabine for 19 h. Cells were subsequently pre-extracted, fixed and post-labeled with an azide-tagged red fluorescent reporter using click chemistry as detailed in the Materials and Methods. The mean nuclear intensity of the red fluorescent reporter was quantitated by confocal microscopy and is plotted for each cells counted for analysis. “Start” indicates the mean nuclear fluorescence intensity immediately following UVA irradiation. Error bars indicate the standard deviation. Statistics were analyzed by a Student's t test (two-tailed, unpaired). Numbers of cells counted for the analysis are indicated at the top for each data.