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V.T. Ciavatta, E. Trieu, J.H. Boatright, J.M. Nickerson; Effect of Chimeraplast Structure and Damaged Target DNA on Frequency of in vitro Gene Repair . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5199.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Oligonucleotide–mediated gene repair (OGR) shows promise for repairing causative point mutations of ocular genetic diseases. Reports have shown that OGR depends on mismatch repair (MMR) and nucleotide excision repair (NER) proteins and may benefit from transcription–coupled repair (TCR) involvement. The purpose here was to test the hypothesis that changes made to the oligonucleotide–target heteroduplexes so as to mimic structures repaired via MMR, NER, and TCR would increase repair frequencies. Methods: A previously described gene repair assay, in which retinal nuclear extract and repair oligonucleotide can be assessed in vitro for their ability to coordinately repair a point mutation in a plasmid antibiotic resistance gene, was used to test the effect of various oligonucleotide structures and target DNA damage on gene repair frequency. New oligonucleotides were synthesized. The standard oligonucleotide (A) has 10 RNA residues flanking either side of the potential mismatched base and forms a mismatch with the template strand of the target. Oligonucleotide A was amended to contain no RNA residues (B), form a mismatch only with the non–template strand (C), form a mismatch only with the non–template strand and have a free 5'OH 12 bases from the mismatch (D), or have methylated cytosines (E). Plasmid was damaged with nicking enzyme N.BstNBI or formic acid. Oligonucleotide A was used in experiments to examine plasmid damage effects. Results: Oligonucleotide B produced a significantly lower repair frequency than A. Oligonucleotides C and D produced repair frequencies that were not significantly different from A. Plasmid damaged by nicking enzyme was repaired twofold more frequently (219.6 +/– 42.7 TetR/million AmpR) than non–nicked plasmid (109.8 +/– 25.2) (p = 0.047; t–test). Conclusions: Lower B repair frequency confirms that presence of RNA residues in a double–hairpin oligonucleotide enhances gene repair. This emphasizes heteroduplex stability importance and suggests further oligonucleotide changes be aimed at stabilizing this complex. Results for C and D suggest that targeting the non–template strand does not enhance repair. Control of target DNA transcription may be necessary to assess C and D. Increased repair frequency of nicked plasmid further demonstrates similarity of OGR to MMR, suggesting potential for improvement by MMR stimulation.
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