Fluorescent oligonucleotides can serve as suitable alternatives to radiolabeled oligonucleotides.
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.
Ballal
Rahul
R
Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, USA.
Cheema
Amrita
A
Ahmad
Waaqar
W
Rosen
Eliot M
EM
Saha
Tapas
T
D000080242:8-Hydroxy-2'-Deoxyguanosine; D000230:Adenocarcinoma / Q000201:enzymology / Q000473:pathology; D001943:Breast Neoplasms / Q000201:enzymology / Q000473:pathology; D045744:Cell Line, Tumor / Q000201:enzymology; D045647:DNA Glycosylases / Q000378:metabolism; D004260:DNA Repair; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000378:metabolism; D005260:Female; D016650:Fluorescein-5-isothiocyanate / Q000032:analysis* / Q000493:pharmacokinetics; D005456:Fluorescent Dyes / Q000032:analysis* / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D019398:Genes, BRCA1; D006801:Humans; D007553:Isotope Labeling; D009363:Neoplasm Proteins / Q000378:metabolism; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000032:analysis / Q000138:chemical synthesis / Q000378:metabolism; D015345:Oligonucleotide Probes / Q000032:analysis* / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D011868:Radioisotopes / Q000009:adverse effects; D011993:Recombinant Fusion Proteins / Q000502:physiology; D013379:Substrate Specificity; D014162:Transfection; D044767:Ubiquitin-Protein Ligases / Q000502:physiology
D005456:Fluorescent Dyes; D009363:Neoplasm Proteins; D009838:Oligodeoxyribonucleotides; D015345:Oligonucleotide Probes; D011868:Radioisotopes; D011993:Recombinant Fusion Proteins; D000080242:8-Hydroxy-2'-Deoxyguanosine; C111164:BRAP protein, human; D044767:Ubiquitin-Protein Ligases; D045647:DNA Glycosylases; C479058:oxoguanine glycosylase 1, human; D003849:Deoxyguanosine; D016650:Fluorescein-5-isothiocyanate