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Prolonged exposure to radiation from radionuclei used in medical research can cause DNA damage and mutation, which lead to several diseases including cancer. Radioactivity-based experiments are expensive and associated with specialized training, dedication of instruments, approvals, and cleanup with potential hazardous waste. The objective of this study was to find an alternative to the use of radioactivity in medical research using nucleic acid chemistry. FITC-labeled oligonucleotides that contain wild-type (wt) and modified base (8-oxo-G) at the same position and their complementary unlabeled strand were synthesized. Purified DNA repair enzyme, OGG1, and nuclear lysates from MCF-7 breast cancer cells were incubated with double-stranded FITC-labeled wt and 8-oxo-G oligonucleotide to demonstrate the OGG1 incision assay. We found that FITC-coupled oligonucleotides do not impose a steric hindrance during duplex formation, and the fluorescence intensity of the oligonucleotide is comparable with the intensity of the radioactive oligonucleotide. Moreover, we have seen that the OGG1 incision assay can be performed using these fluorescence oligonucleotides, replacing conventional use of radiolabeled oligonucleotides in the assay. Although the use of fluorescent-labeled oligonucleotides was described in detail for incision assays, the technique can be applied to replace a broad range of experiments, where radioactive oligonucleotides are used, eliminating the hazardous consequences of radiation.
Radioactivity has been used extensively in life sciences and medical research as a radiolabel to visualize components or target molecules easily in a biological system. Radioactive sources are also used to study living organisms and to diagnose and treat diseases. Common laboratory practices, including detection of DNA, RNA in polyacrylamide and agarose gels via Southern and Northern blots, EMSA, foot-printing, primer extension, enzyme activity-dependent cleavage/ligation assays, and many other techniques, require the use of radioactive oligonucleotides as probes for the analysis of the experiment performed.1-3 Radioactive nuclei that are in use for these purposes include 32P, 33P, 35S, and 125I. Whichever radioactive nuclei are used, all are biologically hazardous, and precautions must be taken during use. One has to monitor closely his or her own personal radioactive exposure while handling radioactive nuclei, as high or prolonged exposure of radioactivity can cause mutations, DNA damage, and other complications.4,5
In this manuscript, we have demonstrated a novel technique where radioactive oligonucleotides can be replaced by fluorescent oligonucleotides, which in turn is analyzed using any standard fluorescence imager or gel documentation system attached with a CCD camera. Potentially, this technique can be used anywhere that a radiolabeled oligonucleotide is required, with any standard fluorescent label, such as fluorescein (FITC), rhodamine, Cyanine 2 (Cy2), Cy3, Cy5, or Texas Red.
MCF-7, a human mammary adenocarcinoma cell line, was obtained from American Type Culture Collection (Manassas, VA). The cells were cultured in DMEM, supplemented with 10% FBS, as described previously.6
The wild-type BRCA1 expression plasmid (wt BRCA1) was created by cloning human BRCA1 cDNA into pcDNA3 vector (Invitrogen, Carlsbad, CA) using artificially engineered 5′ HindIII and 3′ NotI sites.7 Proliferating cells at 70–80% of confluency were transfected with wt BRCA1 or pcDNA3 (background) expression vector (25 μg plasmid DNA/10 cm cell culture dish) using Lipofectamine 2000 transfection reagent (Invitrogen), according to the manufacturer's protocol, as described previously.8,9
5′ FITC-labeled oligonucleotide was synthesized by Midland Certified Reagent Company (Midland, TX) containing a single, damaged base, 8-oxo-G, or wt guanine (G) at the indicated position (see Table 1). A complementary oligonucleotide that will yield a duplex with wt and 8-oxo-G FITC oligonucleotides was also synthesized with no FITC label (Table 1). Annealing of the oligonucleotides was performed in 1× annealing buffer (10 mM Tris, 100 mM NaCl, 1 mM EDTA). A 1:1 molar mixture of FITC-tagged wt oligonucleotide or 8-oxo-G oligonucleotide was mixed with the same complementary, unlabeled strand and heated up to 90°C for 10 min to disrupt any secondary structures. The oligonucleotide mixtures were slowly annealed to room temperature to allow them to form a perfect duplex, which was used during the incision assay.
OGG1 cleavage activity was performed using a modified oligonucleotide cleavage assay as described previously.10-13 5′ FITC-labeled oligonucleotides were annealed in freshly prepared 1× annealing buffer to obtain wt and 8-oxo-G duplex oligonucleotide. Nuclear extracts were prepared from the experimental samples as described previously.14 Nuclear extracts (100 μg) were incubated with 8-oxo-G or wt 5′ FITC-labeled duplex oligonucleotide in a reaction mixture of 20 μl, containing 2.5 pmol 5′ FITC-labeled double-stranded oligonucleotide, 20 mM HEPES, 5% glycerol, 100 mM NaCl, 5 mM EDTA, 5 mM DTT, 100 μg/ml BSA, and 25 μg/ml polydeoxyinosinic:polydeoxycytidylic acid (pH 7.4), and were incubated for 30–45 min in a 37°C water bath. All reactions were stopped by 0.2% SDS and 0.1 mg/mL proteinase K and heat inactivation at 68°C for 10 min. The DNA was extracted using phenol/chloroform/isoamylalcohol and then mixed with 15 μl loading buffer containing 1× denaturing DNA loading dye (85% formamide and 0.03 N NaOH) and heated at 95°C for 5 min. The products were fractionated through 20% polyacrylamide gels containing 7 M urea at 300 volts for 70–80 min. The gels were washed in water and imaged via Ettan DIGE fluorescence imager using the Cy2 filter (GE Healthcare, Piscataway, NJ). The gel images were analyzed using National Institutes of Health-ImageJ software, Version 1.38× (http://rsbweb.nih.gov/ij/). The product bands were quantified relative to the sum of the substrate and product bands and expressed in percentage. At least three independent experiments and t-tests for statistical analysis were performed.
Various amounts of FITC-labeled 8-oxo-G oligonucleotide (purified and unpurified) were loaded in a 20% denaturing PAGE containing 7 M urea. A gradual increase in the fluorescence intensity of the single-stranded 8-oxo-G oligonucleotide band corresponding to a 33-mer appears with increasing concentration (Fig. 1. A). The last two lanes correspond to the purified 8-oxo-G FITC oligonucleotide. Purification was performed using a G-25 mini-column (GE Healthcare), according to the manufacturer's instruction. The G-25 column removes the free, unincorporated FITC molecules. Thus, the fluorescence-labeled single-stranded oligonucleotide can be detected easily using a fluorescence imager.
Next, we wanted to see whether the FITC label could create any problems during duplex formation. We prepared the duplex oligonucleotides with 8-oxo-G oligonucleotide and its complementary, unlabeled strand as described in Materials and Methods. The double-stranded oligonucleotide was then fractionated in a 20% native PAGE. The large size of the fluorophore (in comparison with radioactive nuclei) did not interfere with the duplex formation (Fig. 1B). In both cases, PAGE gels were imaged directly via a Cy2 filter on an Ettan DIGE fluorescence imager (GE Healthcare).
For comparison, the same oligonucleotide was labeled with γ32P-ATP at the 5′ end and subsequently run on a 20% denaturing urea PAGE. The gel was dried and exposed for 6 h and an autoradiograph obtained. There is no visible difference in the detection of the oligonucleotide band intensity for both methods (compare Fig. 1A with C).
8-oxo-G lesions originate in the genomic DNA during oxidative stress, and such DNA becomes the substrate of the DNA repair enzyme OGG1, which helps remove the 8-oxo-G lesion from the genomic DNA by cleaving it at that position.15,16 The specificity of the FITC-labeled oligonucleotides was assessed using the purified OGG1 enzyme (Trivegen, Gaithersburg, MD). wt and 8-oxo-G duplex oligonucleotides were incubated with purified OGG1, and cleavage was analyzed after 30 min. OGG1 cleaved the 8-oxo-G oligonucleotide at the modified base, thus generating a 17-mer FITC nucleotide from the full-length of 33 nucleotides (Fig. 2. A). The wt oligo does not contain an 8-oxo-G lesion and was not a substrate for OGG1 (Fig. 2A).
The FITC-labeled oligonucleotides were used to determine the OGG1 enzyme activity in breast cancer cell line (MCF-7) extract. Nuclear lysates from nontransfected MCF-7 cells and MCF-7 cells, transiently transfected with background vector (pcDNA3) or wt BRCA1, were incubated with wt and 8-oxo-G duplex oligonucleotide to determine the OGG1 incision activity in nuclear lysates.3,10,17 Breast cancer-susceptible gene 1 (BRCA1) was first identified based on its linkage to hereditary breast and ovarian cancers.18 Since the discovery of the gene, the protein has been shown to be involved in a number of cellular processes.19-26 The cleaved products were separated in 20% urea PAGE and imaged in DIGE fluorescence imager. Incision activity of OGG1 from the nuclear lysate is only observed with 8-oxo-G oligonucleotides. Moreover, lysates from BRCA1 over expressed cells show enhanced cleavage activity when compared with background vector pcDNA3 and vehicle (Fig. 2B). Densitometry analysis of the cleavage activity is shown in Figure 2C.
Radioactivity-based experiments are expensive and associated with heavy cleanup with potential hazardous waste. Moreover, a considerable amount of time has to be dedicated for an individual's radioactive training and use approvals by the institution/university, as well as designation of work place and equipment. Thus, an alternative to radioactivity is highly desirable. The most common trend is to use biotinylated oligonucleotides. Biotinylation requires the use of expensive antibodies for detection, prolonging the experiment, and at the same time, using other expensive chemical reagents.
Fluorescent-tagged oligonucleotides can be used efficiently to replace the conventional radioactive, nuclei-tagged oligonucleotides. Fluorescence oligonucleotides are available prepared, purified, and ready to use, making them a convenient alternative. In contrast, radioactive oligonucleotides have to be prepared prior to use via enzymatic coupling of radioactive nuclei to the single-stranded oligonucleotide by T4 polynucleotide kinase or Klenow large fragment.27,28 Furthermore, gels containing radioactive nucleotides need to be dried prior to exposure to an X-ray film to obtain a sharp image in the autoradiogram. These extra steps prolong the experiments and produce a recurring cost of purchasing films, fixer, and developer. However, with a fluorescent label, the gel can be analyzed immediately with an imager, without any prior requirement of drying (Figs. 1 and and2).2). Even the scanning of the gels containing fluorescent oligonucleotide and the subsequent analysis can be performed repeatedly over 3–4 months, as intensity of the fluorescent label does not decrease over time, provided the gel remained in moist condition. Thus, the synthesized fluorescent oligonucleotide can be stored and used any time for the same or different experiments. The only advantage to radioactive labels over fluorescent labels is the size of the label. Radioactive nuclei are small as compared with fluorescent labels, thus a possible steric hindrance might occur with fluorescent labels, although we did not observe it in our experiments.
Although the use of fluorescent-labeled oligonucleotides was described in detail here for incision assays, the technique can be applied to replace a broad range of radioactivity experiments. The wide range of applications of this technique is certainly invaluable, and if used properly, any laboratory can reduce waste, costs, and time of experimentation.
T. S. is thankful to the American Cancer Society (IRG #97-152-16-2), the Fisher Center for Familial Cancer, Georgetown University, and United States Public Health Service Grant RO1-CA80000 (E. M. R.) for financial support.