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Vincent Ciavatta, Priscila Cunha, Jeffrey Boatright, Sophia Tang; A Non-Radioactive Assay for Measuring Retinal Base Excision Repair Capability. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2470.
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© ARVO (1962-2015); The Authors (2016-present)
We are attempting to use endogenous retinal DNA repair capability as part of a novel gene therapy approach. We aim to enhance DNA repair capability through physiological and pharmacological means. Oxoguanine glycosylase (OGG1) is an enzyme needed for repairing oxidized guanine residues, and it is expressed in several mammalian retina cell types. To assess the impact of our efforts, we developed a relatively inexpensive, fluorescence-based, non-radioactive assay to quantify OGG1 enzyme activity.
Methods - For substrate, a 50-base oligonucleotide with an 8-oxodG residue at nucleotide (nt) position 26 and a fluorescent moiety at the 5’ end was annealed to its complementary strand. Annealing (100 fmol/µL each oligonucleotide) was done according to Lan et al., 2003. Protein extracts were prepared from fresh frozen C57BL6 mouse cortex and retina from C57BL6 and RD10 mice according to Bigot et al., 2009 and stored at -80°C. Cut reactions were performed at 32°C for 1 h in 50 or 100 µL using 250 fmol double-stranded target, 5 to 100 µg protein extract, 50 mM HEPES (pH 7.6), 2 mM EDTA, 50 mM NaCl, 5% glycerol, and 0.1 mg/mL BSA. Reactions were stopped with 0.1 M NaOH and 37°C for 15 min. DNA was recovered by ethanol precipitation, dissolved in 90% formamide, heat denatured, resolved by 7M urea, 15% PAGE, and DNA bands were photographed, digitized, and quantified.
Intensity of a 24 nt band showed a dose dependent relationship with amount of retinal protein added up to 100 µg. The diagnostic 24 nt band was detected in all retina and brain samples when using the lowest amount of protein (5 µg). Omitting protein produced no detectable 24 nt band. Product was nearly eliminated if 0.1 M NaOH was not used to stop the reaction. In weanling-aged mice, OGG1 activity was greater in C57BL6 than rd10 retina.
This non-radioactive OGG1 assay is a unique refinement of one established OGG1 activity assay that uses radiolabeled substrates and another fluorescence-based method using a hairpin, single-stranded oligonucleotide. Assay sensitivity approximates that from the established methods. The assay is amenable to high throughput, fluorescence-based detection systems and is useful for measuring effects of various independent variables on retinal OGG1 activity. Retinas undergoing degeneration may have less DNA repair capability than wildtype retinas.
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