Article pubs.acs.org/ac
Detection of UV-Induced Mutagenic Thymine Dimer Using Graphene Oxide Chan Ho Chung,†,‡,∥ Joong Hyun Kim,§,∥ and Bong Hyun Chung*,†,‡ †
BioNano Health Guard Research Center, Korea Research Institute of Bioscience and Biotechnology, 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806, South Korea ‡ Nanobiotechnology, University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon 305-333, South Korea § Medical Device Development Center, Daegu-Gyeongbuk Medical Innovation Foundation, 80 Cheombok-ro, Dong-gu, Daegu 701-310, South Korea S Supporting Information *
ABSTRACT: In this paper, we report for the first time that graphene oxide (GO) can interact with mutagenic DNA but not intact DNA. After UV-irradiated fluorophore-linked DNA containing thymine repeats was mixed with GO, a decrease in fluorescence was observed in a timedependent manner. In contrast, no fluorescence change was observed with intact DNA, indicating that UV irradiation of DNA resulted in the formation of mutagenic bases. Because GO is known to act as a fluorescence quencher, the decreased fluorescence implies adsorption of the UV-irradiated DNA onto GO. It appears that the decreased fluorescence might result from the greater accessibility of hydrophobic methyl groups and phenyl rings of thymine dimers to GO and from deformed DNA structures with less effective charge shielding under saltcontaining conditions. Using this affinity of GO for mutagenic DNA, we could detect UV-irradiated DNA at concentrations as low as 100 pM. We were also able to analyze the ability of phototoxic drugs to catalyze the formation of mutagens under UV irradiation with GO. Because our method is highly sensitive and feasible and does not require the pretreatment of DNA, we propose that it could accelerate the screening of potential phototoxic drug candidates that would be able to sensitize mutagenic dsDNA. several diseases, including cystic fibrosis, sickle cell anemia, phenylketonuria, and various cancers.8 Therefore, the identification of new binding affinities of graphene with mutated DNA could play an important role in controlling these diseases. Next-generation sequencing methods can detect DNA mutations, but challenges remain in identifying chemical mutations in DNA, including methylation, abasic sites, and photodimers.9,10 In this paper we present the first identification of the affinity of the oxide form of graphene (GO) for mutated nucleobases and its application to the high-throughput screening of the phototoxicity of drugs. In particular, thymine dimers in dsDNA adsorbed onto GO, but intact dsDNA did not. Thymine dimers are the primary causes of skin cancers if they are left unrepaired.11 Mutagenic DNA photoproducts are known to be formed by UV irradiation.12 Even nonsteroidal anti-inflammatory drugs (NSAIDs) potentially catalyze the photochemical production of mutagenic dimers.13 In the United States, skin cancer is the most common form of cancer. More than 3.5 million skin cancers in over 2 million people are diagnosed annually.14 Two-thirds of Australians will have
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etallic nanoparticles, quantum dots, carbon nanotubes, and silica nanoparticles are nanomaterials that have exceptional features, such as small sizes, large surface-to-volume ratios, and customizable chemical, electrical, and optical properties. These nanoparticle properties may allow significant advances in various areas. The interactions between nanomaterials and biomolecules play key roles in the biological applications of sensing, drug delivery, and therapy.1 Recently, graphene, a one-atom-thick layer of graphite, has emerged as a practical alternative to other nanoscaled allotropes, such as carbon nanotubes and fullerene. Graphene also interacts with various biomolecules, including nucleic acids,2 lipids,3 and proteins. 4 In addition, the high water solubility and bioconjugation feasibility of graphene in the oxide form have increased the motivation for its use in biological and medical applications.5 With versatile and significant applications in living organisms, the interactions of graphene with nucleotides are important phenomena to consider in developing nucleic acid handling or analysis tools. Similar to other nanomaterials, such as gold nanoparticles and carbon nanotubes, graphene has a high affinity for single-stranded DNA via noncovalent πstacking interactions. The varying affinity of graphene for nucleobases is also utilized to analyze DNA sequences.6,7 DNA mutation is considered to be a primary biological cause of © XXXX American Chemical Society
Received: May 16, 2014 Accepted: November 6, 2014
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cancer before the age of 70.15 Despite their medical importance, current analytical methods to detect mutated bases have limitations for high-throughput analysis. In previous studies, we identified the desorption of thymine dimer-derived structural deformation in ssDNA from gold nanoparticles and the saltinduced aggregation of the particles.16 These interactions allowed the colorimetric detection of the dimer and the highthroughput analysis of drug genetic toxicity. However, gold nanoparticles are not stable in salt-containing buffer and do not interact with dsDNA. Therefore, the colorimetric method may not detect the phenomena under physiological conditions. In contrast, salt-stable GO may be a better candidate for the detection of mutated DNA under physiological conditions. To monitor the behavior of graphene with the photochemically mutated DNA, the fluorescence intensity was measured in samples containing UV-irradiated dsDNA and GO (Scheme 1).
tained from Bioneer Corp. (Daejeon, Korea). Anti cyclobutane pyrimidine dimer (anti-CPD) antibody (clone TDM2 antibody) and other antibodies were obtained from Cosmo Bio Co., Ltd. (Japan). GO Synthesis. GO was synthesized using a modified Hummers method.17 Briefly, a mixture of 3 g of graphite flakes and 1.5 g of NaNO3 was dissolved in 69 mL of concentrated H2SO4 and the resulting solution cooled to 0 °C. Then 9 g of KMnO4 was slowly added to the solution, maintaining the temperature below 20 °C. After the solution was stirred for 30 min, 138 mL of distilled water was slowly added, and the temperature rose to 98 °C due to an exothermic reaction. The solution was stirred and then diluted with 420 mL of water and 3 mL of 30% H2O2. The resultant solution was washed and filtered until the pH was neutral. Dialysis was performed to remove excess ions. The stock solution, containing 2 mg/mL GO, was used after appropriate dilution. Fluorescence Detection of the UV-Induced DNA Photodimers and Screening of Photosensitizing Drugs. DNA samples were purged with argon gas for 30 min and placed in a sealed quartz cuvette or in 96-well plates. A UV lamp (6 W/cm2, 302 nm, Cole-Parmer, United States) was used for irradiation. After exposure to UV irradiation for varying time durations, the fluorescence intensity was measured. For the salt-dependent adsorption experiments, 25 mM HEPES buffer (pH 7.6) with or without 40 mM NaCl was used at 25 °C. To screen the photosensitizing drugs, DNA samples were mixed with a 20 μM concentration of each drug, irradiated under the UV lamp for 30 min, and analyzed as described above. Anti-CPD Assay. DNA samples were prepared as described above by being purged with argon gas and exposed to UV radiation for various lengths of time. DNA was added to poly(vinyl chloride) flat-bottom microtiter plates that had been precoated with protamine sulfate. After being dried overnight at 42 °C, the plates were washed with PBS-T (0.05% Tween-20 in phosphate-buffered saline (PBS)) and incubated with a blocking solution of 2% fetal bovine serum (FBS) in PBS to avoid nonspecific antibody binding. The plates were sequentially incubated with TDM-2 antibodies specific for CPD, biotin-labeled secondary antibody, and streptavidin conjugated to horseradish peroxidase (HRP). The plates were washed with PBS-T at each step. For detection, a substrate solution containing o-phenylenediamine and H2O2 in citrate−phosphate buffer (pH 5.0) was added to the plates, and the plates were incubated for 30 min at 37 °C. Then 2 M H2SO4 was added to the plates, and the absorbance derived from CPD was measured at 492 nm using a microplate reader. DNA Melting Temperature Assay. For determining the melting temperature, the fluorescently labeled DNA and the quencher-labeled DNA were mixed for hybridization in a 2× saline−sodium citrate (SSC), 0.1 M Tris buffer at pH 7.0 and incubated overnight. After removal of unbound oligonucleotides by the addition of a 0.2 mg/mL GO solution and centrifugation, the solution was placed in polymerase chain reaction (PCR) tubes (Bio-Rad) for real-time PCR in a thermal cycler (CFX96, Bio-Rad) and subjected to a temperature gradient. After stabilization for 10 min at 20 °C, the temperature was increased from 20 to 60 °C at a rate of 1 °C/5 min, and fluorescence was measured every 5 min. The melting temperature of the DNA was defined as the inflection point of the sigmoid curve when the relative increasing fluorescence was plotted versus the temperature. The relative
Scheme 1. Interactions of GO with Mutagenic DNAa
a If GO has a specific affinity for the mutagenic thymine dimers that are formed as a result of UV irradiation of DNA, the fluorescence of the DNA-linked fluorophore could be quenched by fluorescence transfer from the donor to GO. Therefore, the affinity between GO and the thymine dimers in DNA could be investigated by measuring the fluorescence change of UV-irradiated DNA after it is mixed with GO.
Because GO acts as a fluorescence quencher, the adsorption or desorption of GO to DNA linked to fluorescent donor molecules was monitored by measuring the fluorescence of the GO−DNA mixture.
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MATERIALS AND METHODS Chemicals and Oligonucleotides. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) and used as received. The oligonucleotides poly(dA)20 (5′-AAAAAAAAAAAAAAAAAAAA-3′), fluorescence-modified poly(dT)20 (5′Alexa Fluor 488 dye-TTTTTTTTTTTTTTTTTTTT-3′), quencher-modified poly(dA)20 (5′-AAAAAAAAAAAAAAAAAAAA-Dabcyl quencher-3′), triple TT (5′-TTAGCATTGCGATTGCATGA-3′-dye) and its complementary sequence (5′TCATGCAATCGCAATGCTAA-3′), intra (5′-AGCATGCGATTGCATGATAC-3′-dye) and its complementary sequence (5′-GTATCATGCAATCGCATGCT-3′), end (5′TTAGCATGCGAGCATGATAC-3′-dye) and its complementary sequence (5′-GTATCATGCTCGCATGCTAA-3′), negative control (5′-AGCATGCGAGCATGATACGT-3′-dye) and its complementary sequence (5′-ACGTATCATGCTCGCATGCT-3′), and other oligonucleotides were obB
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fluorescence was defined as the ratio of the fluorescence at a given temperature relative to the fluorescence at the starting temperature.
formation occurs via covalent linkages between adjacent thymines or cytosines in the same DNA strand. The formation of photoproducts in DNA causes structural deformations of DNA such as kicks and bends.21,22 We postulated that the thymine dimer formed by UV irradiation deformed the DNA structure and exposed hydrophobic groups to GO. Then GO adsorbed the UV-irradiated DNA through hydrophobic interactions. In addition, the interaction of GO with UVirradiated dsDNA was tested, similar to that of ssDNA under high-salt conditions. After the fluorescence-modified poly(dT)20 and its complementary sequence, poly(dA)20, were annealed, the dsDNA was treated with GO to remove trace amounts of ssDNA. Similar to that of the ssDNA, a decreased fluorescence intensity from UV-irradiated poly(dT·dA)20 was observed. The fluorescence intensity decreased as the dsDNA UV irradiation time increased, reaching saturation after 120 min (Figure 2). The decreased florescence was not likely the result
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RESULTS AND DISCUSSION As previously reported, almost 100% of the fluorescence was quenched in the GO−ssDNA mixture in the high-salt conditions (Figure 1). The quenched fluorescence indicates
Figure 1. DNA adsorption on the surface of GO after UV irradiation and at various salt concentrations.
that most of the ssDNA was adsorbed onto GO.18 The adsorption of DNA onto GO may be controlled by multiple factors, including the concentration of cations, the pH, and the presence of organic solvents. As shown in Figure 1, under lowsalt conditions, there was a slight decrease of fluorescence intensity between the mixtures and control. This result indicates that the binding affinity of ssDNA on GO was quite low under low-salt conditions. Significant fluorescence quenching was observed in the mixture of UV-irradiated ssDNA and GO under low-salt conditions. It appears that the decreased fluorescence might have been caused by UV irradiation-driven photobleaching. However, no photobleaching effects of UV irradiation were observed during the evaluated time (Figure S1, Supporting Information). Therefore, the decreased fluorescence of UV-irradiated ssDNA after it was mixed with GO was the result of affinity changes of the ssDNA containing mutated photoproducts. As shown in a previous study, GO is known to be highly negatively charged in deionized (DI) water, resulting in stable aqueous GO suspensions due to the large electrostatic repulsion between GO molecules.19 At low concentrations of NaCl (up to 40 mM), the hydrodynamic diameter (Dh) was fairly constant at approximately 530 nm. As the NaCl concentration increased to 100 mM, the hydrodynamic diameter of GO increased from 531.6 ± 9.18 to 2447.7 ± 144.09 nm (Figure S2, Supporting Information). Therefore, all of the experiments in this study were carried out with concentrations of less than 40 mM NaCl so that they were not affected by GO aggregation. A previous study indicated that the orientation of thymine on gold surfaces or gold nanoparticles is altered so that hydrophobic phenyl rings and methyl groups are exposed to the water phase, thereby creating hydrophobic surfaces of gold nanoparticles.20 Among several products, cis-syn-CPDs are the most frequently formed. This
Figure 2. Fluorescence spectrum of the mixture of GO and 100 nM double-stranded DNA (poly(dA)20·fluorescence-modified poly(dT)20) depending on the UV exposure time.
of photobleaching from the UV irradiation of fluorophores linked to the DNA. We further investigated the sensitivity of GO for the detection of thymine dimers in dsDNA using various concentrations of GO and oligonucleotides. As shown in Figure S3 (Supporting Information), 0.1 mg/mL GO could detect concentrations of UV-irradiated dsDNA as low as 100 pM. This sensitivity is 200-fold greater than that of gold nanoparticles.23 To confirm whether UV irradiation led to the photosensitive activity in oligonucleotides that resulted in CPD formation, we measured the degrees of CPD formation by using various DNA sequences with different number of two adjacent thymines. The oligonucleotide sequences used in this study were the same in length but different in the number or location of their thymine−thymine content. We found a simple correlation between the number of two adjacent thymines and the number of dimers formed after 2 h of irradiation. As shown in Figure 3, the relative fluorescence change increased with an increase in the number of thymine−thymine pairs. The photochemical reaction did not preferentially occur at the end or middle of the strands as previously reported.24 Among the tested sequences, a random sequence as a control showed a relatively low level of fluorescence decrease as the UV irradiation time increased. It has been shown that two nonsuccessively located thymines could form dimers as a result of interstrand-type reactions.25 The fluorescence changes were dependent on the number of successive thymines as well as the UV irradiation time. These C
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Figure 3. Time-dependent adsorption kinetics of UV exposure on various types of DNA: poly(dT)20, triple TT, intra, end, and negative control. F0 and F indicate the fluorescence intensity of the control and that after each duration of UV exposure, respectively.
Figure 5. Melting temperature analysis of UV-irradiated dsDNA.
room temperature. These results confirmed that there was no significant dehybridization of the UV-irradiated dsDNA. UV irradiation of dsDNA might result in the formation of covalent bonds between adjacent thymines. A greater number of thymine dimers could be formed with increasing irradiation time, resulting in decreased fluorescence due to more adsorption of the UV-irradiated dsDNA. Structure- and basedependent interactions of DNA on gold nanoparticles28 and carbon nanotubes27 have been previously reported. This paper is the first to describe the interactions of GO with thymine dimers in both single- and double-stranded DNA. We applied the mutagenic DNA-interacting properties of GO to a high-throughput analysis of drug phototoxicity. For analyzing the phototoxicity of drugs, UV-irradiated mixtures of DNA and each of the selected drugs were mixed with GO, and the fluorescence change of the mixture was monitored. Three NSAIDs [ketoprofen (KP), naproxen (NP), and indomethacin (IM)] and two carbonyl derivatives [acetophenone (AP) and benzophenone (BP)] were tested. As shown in Figure 6, the fluorescence intensity decreased when dsDNA was irradiated in the presence of AP, BP, and KP, which are well-known photocatalyzing drugs. However, NP and IM did not markedly alter the fluorescence of the mixtures, indicating their smaller photosensitizing activities. The photocatalyzing degree was converted to the fluorescence quantum yield (i.e., the fluorescence ratio divided by the absorbance of the drug). This result also agreed with those of previous studies29,30 on the mutation-photocatalyzing effects of the drugs.
results confirm the formation of CPDs and their adsorption onto GO. Although our method has many advantages over other previous methods, it still has some limitations so that the specific region of the photodimer in the strand with thymine cannot be determined. One other possible explanation is that the UV-irradiated poly(dT·dA)20 was dehybridized into ssDNAs and adsorbed onto GO. The dehybridization of poly(dT·dA)20 from UV irradiation was investigated using an enzyme-linked immunosorbent assay (ELISA) with a CPD-specific antibody and using Tm analysis. In the ELISA, the absorbance intensity from dsDNA was less than that from ssDNA (Figure 4). The anti-
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CONCLUSIONS In conclusion, we report for the first time the affinity of GO for mutagenic thymine dimers in DNA and the application of GO in the high-throughput analysis of photosensitizing drugs. In previous studies, the desorption of ssDNA containing thymine dimers from gold nanoparticles was driven by a conformational change.27 Under low-salt conditions, negatively charged phosphates in DNA are not shielded enough to allow hydrophobic π-stacking interactions between GO and DNA. These interactions are the dominant forces under high-salt conditions. If thymine bases in DNA are exposed to sufficient light energy, the adjacent bases can covalently bond with each other, resulting in severe widening of the minor and major DNA grooves, as well as the kick and bend of the DNA structure.21,22 In this deformed structure, the flat GO surface could interact with the hydrophobic phenyl rings and methyl
Figure 4. Measurements of UV-induced DNA damage in single- and double-stranded DNA by CPD ELISA. Absorbance at 492 nm (top) and photograph of the plate (bottom).
CPD antibody used in the ELISA binds to CPDs formed in every dipyrimidine sequence in single-stranded DNA but binds to CPD in dsDNA to a lesser extent.26 This result implies that a majority of the UV-irradiated DNA formed double strands. According to the Tm analysis of the dsDNA in Figure 5 and Table S1 (Supporting Information), the Tm of the dsDNA exposed to UV light decreased in a time-dependent manner. Additionally, the Tm of dsDNA exposed to UV light for 120 min was found to be 14.4 °C, higher than that of the control at D
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AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Fax: +82-42-879-8594. Author Contributions ∥
C.H.C. and J.H.K. contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the BioNano Health Guard Research Center funded by the Ministry of Science, ICT & Future Planning (MSIP) of Korea as a Global Frontier Project (Grant H-GUARD_2013M3A6B2078950) and the KRIBB Initiative Program, Republic of Korea.
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Figure 6. Screening of the phototoxicity of NSAIDs with GO. (a) Fluorescence intensity of UV-irradiated samples in the absence and presence of photosensitizing drugs. (b) Flourimetric quantum yield (QY) of the drugs for photosensitizing thymine dimers. The flourimetric quantum yields of the compounds to photosensitize dimer formation were defined by by dividing the relative fluorescence change of the tested samples to the fluorescence of the control ((F0 − F)/F0)) into absorbance of the tested compounds at 302 nm. F0 and F are the fluorescence intensity of the control and that after each time of UV exposure, respectively.
groups of the thymine dimers. The stability of GO in saltcontaining buffers allows investigation of its interactions with the mutagenic bases. As gold nanoparticles are unstable at highsalt concentrations, investigating these interactions with gold nanoparticles is difficult in salt-containing solutions. Salt plays an important role in the interaction between GO and the dimers. The adsorption of mutagenic bases in DNA might be less effective in the absence of salt because no shielding of the negatively charged phosphates in DNA is expected. We are currently investigating the more profound effect of salt on this interaction and on cytosine dimers and the number of hydroxyl groups on the GO surface. Decoding the base information in DNA is extremely important for predicting, detecting, and controlling gene-related diseases and also for developing drugs. To that end, the development of a novel DNA sequencing technology has been the subject of recent research. Obtaining such information on the chemical interactions of GO with DNA may allow the gene sequencing of normal DNA and mutagenic DNA and may aid in the development of new therapeutic drugs.
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ASSOCIATED CONTENT
S Supporting Information *
Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. E
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(27) Hiromi, K.-S.; Dmitri, Y. P.; Michael, J. T.; Lloyd, J. W. J. Am. Chem. Soc. 2003, 125, 9014−9015. (28) Fernando, A.; Mary, E. H.; Jene, A. G.; Daniel, B. Nanotechnology 2009, 20, 395101. (29) Douki, T.; Reynaud-Angelin, A.; Cadet, J.; Sage, E. Biochemistry 2003, 42, 9221−9226. (30) Nadia, C.-L.; Martine, D.; Nicole, P. Biochem. Pharmacol. 1998, 55, 441−446.
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Detection of UV-Induced Mutagenic Thymine Dimer Using Graphene Oxide Chan Ho Chung,†,‡,∥ Joong Hyun Kim,§,∥ and Bong Hyun Chung*,†,‡ †
BioNano Health Guard Research Center, Korea Research Institute of Bioscience and Biotechnology, 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806, South Korea ‡ Nanobiotechnology, University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon 305-333, South Korea § Medical Device Development Center, Daegu-Gyeongbuk Medical Innovation Foundation, 80 Cheombok-ro, Dong-gu, Daegu 701-310, South Korea S Supporting Information *
ABSTRACT: In this paper, we report for the first time that graphene oxide (GO) can interact with mutagenic DNA but not intact DNA. After UV-irradiated fluorophore-linked DNA containing thymine repeats was mixed with GO, a decrease in fluorescence was observed in a timedependent manner. In contrast, no fluorescence change was observed with intact DNA, indicating that UV irradiation of DNA resulted in the formation of mutagenic bases. Because GO is known to act as a fluorescence quencher, the decreased fluorescence implies adsorption of the UV-irradiated DNA onto GO. It appears that the decreased fluorescence might result from the greater accessibility of hydrophobic methyl groups and phenyl rings of thymine dimers to GO and from deformed DNA structures with less effective charge shielding under saltcontaining conditions. Using this affinity of GO for mutagenic DNA, we could detect UV-irradiated DNA at concentrations as low as 100 pM. We were also able to analyze the ability of phototoxic drugs to catalyze the formation of mutagens under UV irradiation with GO. Because our method is highly sensitive and feasible and does not require the pretreatment of DNA, we propose that it could accelerate the screening of potential phototoxic drug candidates that would be able to sensitize mutagenic dsDNA. several diseases, including cystic fibrosis, sickle cell anemia, phenylketonuria, and various cancers.8 Therefore, the identification of new binding affinities of graphene with mutated DNA could play an important role in controlling these diseases. Next-generation sequencing methods can detect DNA mutations, but challenges remain in identifying chemical mutations in DNA, including methylation, abasic sites, and photodimers.9,10 In this paper we present the first identification of the affinity of the oxide form of graphene (GO) for mutated nucleobases and its application to the high-throughput screening of the phototoxicity of drugs. In particular, thymine dimers in dsDNA adsorbed onto GO, but intact dsDNA did not. Thymine dimers are the primary causes of skin cancers if they are left unrepaired.11 Mutagenic DNA photoproducts are known to be formed by UV irradiation.12 Even nonsteroidal anti-inflammatory drugs (NSAIDs) potentially catalyze the photochemical production of mutagenic dimers.13 In the United States, skin cancer is the most common form of cancer. More than 3.5 million skin cancers in over 2 million people are diagnosed annually.14 Two-thirds of Australians will have
M
etallic nanoparticles, quantum dots, carbon nanotubes, and silica nanoparticles are nanomaterials that have exceptional features, such as small sizes, large surface-to-volume ratios, and customizable chemical, electrical, and optical properties. These nanoparticle properties may allow significant advances in various areas. The interactions between nanomaterials and biomolecules play key roles in the biological applications of sensing, drug delivery, and therapy.1 Recently, graphene, a one-atom-thick layer of graphite, has emerged as a practical alternative to other nanoscaled allotropes, such as carbon nanotubes and fullerene. Graphene also interacts with various biomolecules, including nucleic acids,2 lipids,3 and proteins. 4 In addition, the high water solubility and bioconjugation feasibility of graphene in the oxide form have increased the motivation for its use in biological and medical applications.5 With versatile and significant applications in living organisms, the interactions of graphene with nucleotides are important phenomena to consider in developing nucleic acid handling or analysis tools. Similar to other nanomaterials, such as gold nanoparticles and carbon nanotubes, graphene has a high affinity for single-stranded DNA via noncovalent πstacking interactions. The varying affinity of graphene for nucleobases is also utilized to analyze DNA sequences.6,7 DNA mutation is considered to be a primary biological cause of © XXXX American Chemical Society
Received: May 16, 2014 Accepted: November 6, 2014
A
dx.doi.org/10.1021/ac503577t | Anal. Chem. XXXX, XXX, XXX−XXX
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cancer before the age of 70.15 Despite their medical importance, current analytical methods to detect mutated bases have limitations for high-throughput analysis. In previous studies, we identified the desorption of thymine dimer-derived structural deformation in ssDNA from gold nanoparticles and the saltinduced aggregation of the particles.16 These interactions allowed the colorimetric detection of the dimer and the highthroughput analysis of drug genetic toxicity. However, gold nanoparticles are not stable in salt-containing buffer and do not interact with dsDNA. Therefore, the colorimetric method may not detect the phenomena under physiological conditions. In contrast, salt-stable GO may be a better candidate for the detection of mutated DNA under physiological conditions. To monitor the behavior of graphene with the photochemically mutated DNA, the fluorescence intensity was measured in samples containing UV-irradiated dsDNA and GO (Scheme 1).
tained from Bioneer Corp. (Daejeon, Korea). Anti cyclobutane pyrimidine dimer (anti-CPD) antibody (clone TDM2 antibody) and other antibodies were obtained from Cosmo Bio Co., Ltd. (Japan). GO Synthesis. GO was synthesized using a modified Hummers method.17 Briefly, a mixture of 3 g of graphite flakes and 1.5 g of NaNO3 was dissolved in 69 mL of concentrated H2SO4 and the resulting solution cooled to 0 °C. Then 9 g of KMnO4 was slowly added to the solution, maintaining the temperature below 20 °C. After the solution was stirred for 30 min, 138 mL of distilled water was slowly added, and the temperature rose to 98 °C due to an exothermic reaction. The solution was stirred and then diluted with 420 mL of water and 3 mL of 30% H2O2. The resultant solution was washed and filtered until the pH was neutral. Dialysis was performed to remove excess ions. The stock solution, containing 2 mg/mL GO, was used after appropriate dilution. Fluorescence Detection of the UV-Induced DNA Photodimers and Screening of Photosensitizing Drugs. DNA samples were purged with argon gas for 30 min and placed in a sealed quartz cuvette or in 96-well plates. A UV lamp (6 W/cm2, 302 nm, Cole-Parmer, United States) was used for irradiation. After exposure to UV irradiation for varying time durations, the fluorescence intensity was measured. For the salt-dependent adsorption experiments, 25 mM HEPES buffer (pH 7.6) with or without 40 mM NaCl was used at 25 °C. To screen the photosensitizing drugs, DNA samples were mixed with a 20 μM concentration of each drug, irradiated under the UV lamp for 30 min, and analyzed as described above. Anti-CPD Assay. DNA samples were prepared as described above by being purged with argon gas and exposed to UV radiation for various lengths of time. DNA was added to poly(vinyl chloride) flat-bottom microtiter plates that had been precoated with protamine sulfate. After being dried overnight at 42 °C, the plates were washed with PBS-T (0.05% Tween-20 in phosphate-buffered saline (PBS)) and incubated with a blocking solution of 2% fetal bovine serum (FBS) in PBS to avoid nonspecific antibody binding. The plates were sequentially incubated with TDM-2 antibodies specific for CPD, biotin-labeled secondary antibody, and streptavidin conjugated to horseradish peroxidase (HRP). The plates were washed with PBS-T at each step. For detection, a substrate solution containing o-phenylenediamine and H2O2 in citrate−phosphate buffer (pH 5.0) was added to the plates, and the plates were incubated for 30 min at 37 °C. Then 2 M H2SO4 was added to the plates, and the absorbance derived from CPD was measured at 492 nm using a microplate reader. DNA Melting Temperature Assay. For determining the melting temperature, the fluorescently labeled DNA and the quencher-labeled DNA were mixed for hybridization in a 2× saline−sodium citrate (SSC), 0.1 M Tris buffer at pH 7.0 and incubated overnight. After removal of unbound oligonucleotides by the addition of a 0.2 mg/mL GO solution and centrifugation, the solution was placed in polymerase chain reaction (PCR) tubes (Bio-Rad) for real-time PCR in a thermal cycler (CFX96, Bio-Rad) and subjected to a temperature gradient. After stabilization for 10 min at 20 °C, the temperature was increased from 20 to 60 °C at a rate of 1 °C/5 min, and fluorescence was measured every 5 min. The melting temperature of the DNA was defined as the inflection point of the sigmoid curve when the relative increasing fluorescence was plotted versus the temperature. The relative
Scheme 1. Interactions of GO with Mutagenic DNAa
a If GO has a specific affinity for the mutagenic thymine dimers that are formed as a result of UV irradiation of DNA, the fluorescence of the DNA-linked fluorophore could be quenched by fluorescence transfer from the donor to GO. Therefore, the affinity between GO and the thymine dimers in DNA could be investigated by measuring the fluorescence change of UV-irradiated DNA after it is mixed with GO.
Because GO acts as a fluorescence quencher, the adsorption or desorption of GO to DNA linked to fluorescent donor molecules was monitored by measuring the fluorescence of the GO−DNA mixture.
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MATERIALS AND METHODS Chemicals and Oligonucleotides. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) and used as received. The oligonucleotides poly(dA)20 (5′-AAAAAAAAAAAAAAAAAAAA-3′), fluorescence-modified poly(dT)20 (5′Alexa Fluor 488 dye-TTTTTTTTTTTTTTTTTTTT-3′), quencher-modified poly(dA)20 (5′-AAAAAAAAAAAAAAAAAAAA-Dabcyl quencher-3′), triple TT (5′-TTAGCATTGCGATTGCATGA-3′-dye) and its complementary sequence (5′TCATGCAATCGCAATGCTAA-3′), intra (5′-AGCATGCGATTGCATGATAC-3′-dye) and its complementary sequence (5′-GTATCATGCAATCGCATGCT-3′), end (5′TTAGCATGCGAGCATGATAC-3′-dye) and its complementary sequence (5′-GTATCATGCTCGCATGCTAA-3′), negative control (5′-AGCATGCGAGCATGATACGT-3′-dye) and its complementary sequence (5′-ACGTATCATGCTCGCATGCT-3′), and other oligonucleotides were obB
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fluorescence was defined as the ratio of the fluorescence at a given temperature relative to the fluorescence at the starting temperature.
formation occurs via covalent linkages between adjacent thymines or cytosines in the same DNA strand. The formation of photoproducts in DNA causes structural deformations of DNA such as kicks and bends.21,22 We postulated that the thymine dimer formed by UV irradiation deformed the DNA structure and exposed hydrophobic groups to GO. Then GO adsorbed the UV-irradiated DNA through hydrophobic interactions. In addition, the interaction of GO with UVirradiated dsDNA was tested, similar to that of ssDNA under high-salt conditions. After the fluorescence-modified poly(dT)20 and its complementary sequence, poly(dA)20, were annealed, the dsDNA was treated with GO to remove trace amounts of ssDNA. Similar to that of the ssDNA, a decreased fluorescence intensity from UV-irradiated poly(dT·dA)20 was observed. The fluorescence intensity decreased as the dsDNA UV irradiation time increased, reaching saturation after 120 min (Figure 2). The decreased florescence was not likely the result
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RESULTS AND DISCUSSION As previously reported, almost 100% of the fluorescence was quenched in the GO−ssDNA mixture in the high-salt conditions (Figure 1). The quenched fluorescence indicates
Figure 1. DNA adsorption on the surface of GO after UV irradiation and at various salt concentrations.
that most of the ssDNA was adsorbed onto GO.18 The adsorption of DNA onto GO may be controlled by multiple factors, including the concentration of cations, the pH, and the presence of organic solvents. As shown in Figure 1, under lowsalt conditions, there was a slight decrease of fluorescence intensity between the mixtures and control. This result indicates that the binding affinity of ssDNA on GO was quite low under low-salt conditions. Significant fluorescence quenching was observed in the mixture of UV-irradiated ssDNA and GO under low-salt conditions. It appears that the decreased fluorescence might have been caused by UV irradiation-driven photobleaching. However, no photobleaching effects of UV irradiation were observed during the evaluated time (Figure S1, Supporting Information). Therefore, the decreased fluorescence of UV-irradiated ssDNA after it was mixed with GO was the result of affinity changes of the ssDNA containing mutated photoproducts. As shown in a previous study, GO is known to be highly negatively charged in deionized (DI) water, resulting in stable aqueous GO suspensions due to the large electrostatic repulsion between GO molecules.19 At low concentrations of NaCl (up to 40 mM), the hydrodynamic diameter (Dh) was fairly constant at approximately 530 nm. As the NaCl concentration increased to 100 mM, the hydrodynamic diameter of GO increased from 531.6 ± 9.18 to 2447.7 ± 144.09 nm (Figure S2, Supporting Information). Therefore, all of the experiments in this study were carried out with concentrations of less than 40 mM NaCl so that they were not affected by GO aggregation. A previous study indicated that the orientation of thymine on gold surfaces or gold nanoparticles is altered so that hydrophobic phenyl rings and methyl groups are exposed to the water phase, thereby creating hydrophobic surfaces of gold nanoparticles.20 Among several products, cis-syn-CPDs are the most frequently formed. This
Figure 2. Fluorescence spectrum of the mixture of GO and 100 nM double-stranded DNA (poly(dA)20·fluorescence-modified poly(dT)20) depending on the UV exposure time.
of photobleaching from the UV irradiation of fluorophores linked to the DNA. We further investigated the sensitivity of GO for the detection of thymine dimers in dsDNA using various concentrations of GO and oligonucleotides. As shown in Figure S3 (Supporting Information), 0.1 mg/mL GO could detect concentrations of UV-irradiated dsDNA as low as 100 pM. This sensitivity is 200-fold greater than that of gold nanoparticles.23 To confirm whether UV irradiation led to the photosensitive activity in oligonucleotides that resulted in CPD formation, we measured the degrees of CPD formation by using various DNA sequences with different number of two adjacent thymines. The oligonucleotide sequences used in this study were the same in length but different in the number or location of their thymine−thymine content. We found a simple correlation between the number of two adjacent thymines and the number of dimers formed after 2 h of irradiation. As shown in Figure 3, the relative fluorescence change increased with an increase in the number of thymine−thymine pairs. The photochemical reaction did not preferentially occur at the end or middle of the strands as previously reported.24 Among the tested sequences, a random sequence as a control showed a relatively low level of fluorescence decrease as the UV irradiation time increased. It has been shown that two nonsuccessively located thymines could form dimers as a result of interstrand-type reactions.25 The fluorescence changes were dependent on the number of successive thymines as well as the UV irradiation time. These C
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Figure 3. Time-dependent adsorption kinetics of UV exposure on various types of DNA: poly(dT)20, triple TT, intra, end, and negative control. F0 and F indicate the fluorescence intensity of the control and that after each duration of UV exposure, respectively.
Figure 5. Melting temperature analysis of UV-irradiated dsDNA.
room temperature. These results confirmed that there was no significant dehybridization of the UV-irradiated dsDNA. UV irradiation of dsDNA might result in the formation of covalent bonds between adjacent thymines. A greater number of thymine dimers could be formed with increasing irradiation time, resulting in decreased fluorescence due to more adsorption of the UV-irradiated dsDNA. Structure- and basedependent interactions of DNA on gold nanoparticles28 and carbon nanotubes27 have been previously reported. This paper is the first to describe the interactions of GO with thymine dimers in both single- and double-stranded DNA. We applied the mutagenic DNA-interacting properties of GO to a high-throughput analysis of drug phototoxicity. For analyzing the phototoxicity of drugs, UV-irradiated mixtures of DNA and each of the selected drugs were mixed with GO, and the fluorescence change of the mixture was monitored. Three NSAIDs [ketoprofen (KP), naproxen (NP), and indomethacin (IM)] and two carbonyl derivatives [acetophenone (AP) and benzophenone (BP)] were tested. As shown in Figure 6, the fluorescence intensity decreased when dsDNA was irradiated in the presence of AP, BP, and KP, which are well-known photocatalyzing drugs. However, NP and IM did not markedly alter the fluorescence of the mixtures, indicating their smaller photosensitizing activities. The photocatalyzing degree was converted to the fluorescence quantum yield (i.e., the fluorescence ratio divided by the absorbance of the drug). This result also agreed with those of previous studies29,30 on the mutation-photocatalyzing effects of the drugs.
results confirm the formation of CPDs and their adsorption onto GO. Although our method has many advantages over other previous methods, it still has some limitations so that the specific region of the photodimer in the strand with thymine cannot be determined. One other possible explanation is that the UV-irradiated poly(dT·dA)20 was dehybridized into ssDNAs and adsorbed onto GO. The dehybridization of poly(dT·dA)20 from UV irradiation was investigated using an enzyme-linked immunosorbent assay (ELISA) with a CPD-specific antibody and using Tm analysis. In the ELISA, the absorbance intensity from dsDNA was less than that from ssDNA (Figure 4). The anti-
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CONCLUSIONS In conclusion, we report for the first time the affinity of GO for mutagenic thymine dimers in DNA and the application of GO in the high-throughput analysis of photosensitizing drugs. In previous studies, the desorption of ssDNA containing thymine dimers from gold nanoparticles was driven by a conformational change.27 Under low-salt conditions, negatively charged phosphates in DNA are not shielded enough to allow hydrophobic π-stacking interactions between GO and DNA. These interactions are the dominant forces under high-salt conditions. If thymine bases in DNA are exposed to sufficient light energy, the adjacent bases can covalently bond with each other, resulting in severe widening of the minor and major DNA grooves, as well as the kick and bend of the DNA structure.21,22 In this deformed structure, the flat GO surface could interact with the hydrophobic phenyl rings and methyl
Figure 4. Measurements of UV-induced DNA damage in single- and double-stranded DNA by CPD ELISA. Absorbance at 492 nm (top) and photograph of the plate (bottom).
CPD antibody used in the ELISA binds to CPDs formed in every dipyrimidine sequence in single-stranded DNA but binds to CPD in dsDNA to a lesser extent.26 This result implies that a majority of the UV-irradiated DNA formed double strands. According to the Tm analysis of the dsDNA in Figure 5 and Table S1 (Supporting Information), the Tm of the dsDNA exposed to UV light decreased in a time-dependent manner. Additionally, the Tm of dsDNA exposed to UV light for 120 min was found to be 14.4 °C, higher than that of the control at D
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AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Fax: +82-42-879-8594. Author Contributions ∥
C.H.C. and J.H.K. contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the BioNano Health Guard Research Center funded by the Ministry of Science, ICT & Future Planning (MSIP) of Korea as a Global Frontier Project (Grant H-GUARD_2013M3A6B2078950) and the KRIBB Initiative Program, Republic of Korea.
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Figure 6. Screening of the phototoxicity of NSAIDs with GO. (a) Fluorescence intensity of UV-irradiated samples in the absence and presence of photosensitizing drugs. (b) Flourimetric quantum yield (QY) of the drugs for photosensitizing thymine dimers. The flourimetric quantum yields of the compounds to photosensitize dimer formation were defined by by dividing the relative fluorescence change of the tested samples to the fluorescence of the control ((F0 − F)/F0)) into absorbance of the tested compounds at 302 nm. F0 and F are the fluorescence intensity of the control and that after each time of UV exposure, respectively.
groups of the thymine dimers. The stability of GO in saltcontaining buffers allows investigation of its interactions with the mutagenic bases. As gold nanoparticles are unstable at highsalt concentrations, investigating these interactions with gold nanoparticles is difficult in salt-containing solutions. Salt plays an important role in the interaction between GO and the dimers. The adsorption of mutagenic bases in DNA might be less effective in the absence of salt because no shielding of the negatively charged phosphates in DNA is expected. We are currently investigating the more profound effect of salt on this interaction and on cytosine dimers and the number of hydroxyl groups on the GO surface. Decoding the base information in DNA is extremely important for predicting, detecting, and controlling gene-related diseases and also for developing drugs. To that end, the development of a novel DNA sequencing technology has been the subject of recent research. Obtaining such information on the chemical interactions of GO with DNA may allow the gene sequencing of normal DNA and mutagenic DNA and may aid in the development of new therapeutic drugs.
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ASSOCIATED CONTENT
S Supporting Information *
Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. E
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