- Open Access
Fast and simple method for screening of single-stranded DNA breaking photosensitizers using graphene oxide
© The Author(s) 2018
- Received: 7 September 2018
- Accepted: 24 September 2018
- Published: 22 October 2018
A fast and simple method for screening of ssDNA-breaking photosensitizers was developed using graphene oxide. The ultraviolet light-induced DNA breaks are one of the most harmful DNA damages and cause skin cancer if they are left unrepaired. Since graphene oxide showed relatively strong affinity to the broken DNA than intact DNA, and it quenched fluorescence of the DNA labeling dye effectively, the degree of ultraviolet light-induced broken DNAs could be analyzed by measuring decreased fluorescence after mixing the DNA with graphene oxide. The decrease of fluorescence was highly correlated with the ultraviolet light-irradiating time and concentration of the added drugs. As a result, it was possible to evaluate the efficacy of different ssDNA-breaking photosensitizers in a high-throughput manner. However, conventional methods for the damaged-DNA analysis are time-consuming and require additional manipulations such as purification, radio-labeling, enzymatic digestion, or chemical modification of DNA. The phototoxicity of five drugs such as benzophenone, ketoprofen, indomethacin, naproxen, and norfloxacin was tested using the proposed method. The ssDNA-breaking efficiency of the drugs was well matched with reported efficiency of the tested drugs. In contrast to naked gold nanoparticles, graphene oxide is stably dispersed in the presence of salt, the phototoxicity of the drugs could be successfully tested at a physiological condition using the graphene oxide based method.
- Graphene oxide
- Single-stranded DNA break
- Skin cancer
- Phototoxic drug
In countries with a white population, the most common cancer is skin cancers. Every 10–20 years, incidence of melanoma has doubled in these countries. In 2007, more than 84,500 people in UK was diagnosed with non-melanoma skin cancer . It was reported that about two-thirds of Australian before 70 years old could have skin cancer at least once for their life . Unrepaired photolesions which are formed by ultraviolet (UV) light irradiation is considered as main cause of the skin cancer. The mutated photolesions such as bipyrimidine dimers, apurine sites and strand breaks are produced by adsorption of UV light [3, 4]. Among them, bipyrimidine dimers have the highest frequency than others. However, the other UV light-induced mutants formed to a lesser extent can be more harmful than bipyrimidine dimers . In addition, it is known that occurrence of these types of cellular DNA damages are increased by several drugs due to photosensitization [5–9]. Because of the phototoxicity of the photosensitizers, FDA recommends to provide the photosensitizing effects of drug substances. Comet assay  and Ames test  are representative in vitro genotoxicity tests as standard testing. Beside such cell-based methods, acelluar assays using naked DNA, such as immunological assays , gel electrophoresis , chromatic assays , atomic force microscopy  and electrochemical sensor-based assays , have been also developed to analyze the UV light-induced DNA mutations. However, these approaches are still limited to the high-throughput screening of compounds for phototoxicity because of their dependence on expensive instruments and additional manipulations such as purification, radiolabeling, enzymatic digestion, or chemical modification of the DNA for sensitive and accurate information. In the pharmaceutical industry there have been growing demands for a simplified method to screen for phototoxic chemical compounds in a parallel way, especially a method that can be implemented in an automated system. In previous, a gold nanoparticle (AuNP) based simple colorimetric method to detect the single-stranded (ss)DNA breaks (SDBs) and to screen photosensitizing drugs were reported . However, the AuNP based colorimetric methods are not applicable to a salt containing physiological condition because AuNPs are easily aggregated by salt. However, the colorimetric method may not detect the phenomena under physiological conditions. In contrast, salt-stable dispersion of graphene oxide (GO) could allow its application to detect SDBs in the physiological condition. Due to the selective affinity to ssDNA, and highly effective fluorescence quenching ability, GO has been applied to detect various targets such as heavy metals, small molecules and nucleic acids . Previously, a GO based method to detect DNA photodimers and screen dimer-photosensitizing drugs was reported . However, it is not yet reported if GO could be applied to detect the SDBs and screen photosensitizing drugs. Therefore, in this study, GO was first applied to detect the SDBs and evaluate the photosensitizing effect of drugs under a physiological condition.
All other chemicals except for DNA were purchased from Sigma-Aldrich and used without further purification. The sequence of the DNA used for the detection of SDBs (5′-Texas reds®-GGAAGGTGTGGAAGG-3′) was purchased from Bioneer (Daejeon, Korea). Because the formation of pyrimidine photodimers will interfere with the interaction between ssDNA and GO, special care for designing DNA sequence that included only purines for the specific detection of SDBs was needed . ssDNA was added in a quartz cuvette or a well plate followed by mixing with each drug in 18 µl of 1xPBS (pH 7.4). The DNA sample (100 nM) in the sealed quartz cuvette was irradiated under 25 cm of a UV lamp (15 W, 10,600 µW/cm2 at 312 nm), which was top of a dark box (35 cm × 35 cm × 30 cm) . The irradiated energy to DNA was adjusted by varying the UV irradiation time. For the detection of SDBs, the UV light-irradiated DNA was mixed with GO (2 µg/ml) in PBS for 10 min. For the parallel screening of the photosensitizing drugs, DNA samples were mixed with 20 µM of each drug and irradiated under the UV lamp for 20 min. After 10 min of mixing with GO, fluorescence of the UV light-irradiated samples were recorded using a microplate reader (Infinite M200 Pro, Tecan, Switzerland) with excitation at 560 nm.
In conclusion, herein GO was successfully applied to the screening of drugs for SDB-photosensitizing activity at the physiological condition in an operationally simple and high-throughput manner. In contrast to conventional methods, the GO based method described herein is ideal for the accelerated screening of a large number of drug candidates at the physiological condition because it does not require chemical or biochemical manipulation of the DNA. However, instability of AuNP in the presence of salt limits a practical application of the previously reported colorimetric method for evaluating phototoxicity of the drugs. Therefore, it could be expected that the presented method using GO will provide a feasible and practical approach for developing new drug candidates without phototoxicity.
All authors have contributed to the writing of the manuscript. Both authors read and approved the final manuscript.
The authors declare that they have no competing interests.
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2017R1A2B4006505) and by a Grant from the Next-Generation BioGreen 21 Program (No. PJ01334501), Rural Development Administration, Republic of Korea.
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