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The lack of mutagenic potential of a guanine-rich triplex forming oligonucleotide in physiological conditions

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Title: The lack of mutagenic potential of a guanine-rich triplex forming oligonucleotide in physiological conditions
Authors: Saleh, AF
Fellows, MM
Ying, L
Gooderham, NJ
Priestley, CC
Item Type: Journal Article
Abstract: Triplex forming oligonucleotides (TFOs) bind in the major groove of DNA duplex in a sequence-specific manner imparted by Hoogsteen hydrogen bonds. There have been several reports demonstrating the ability of guanine-rich TFOs to induce targeted mutagenesis on an exogenous plasmid or an endogenous chromosomal locus. In particular, a 30mer guanine-rich triplex forming oligonucleotide, AG30, optimally designed to target the supFG1 reporter gene was reported to be mutagenic in the absence of DNA reactive agents in cultured cells and in vivo Here, we investigated the mutagenic potential of AG30 using the supFG1 shuttle vector forward mutation assay under physiological conditions. We also assessed the triplex binding potential of AG30 alongside cytotoxic and mutagenic assessment. In a cell free condition, AG30 was able to bind its polypurine target site in the supFG1 gene in the absence of potassium chloride and also aligned with a 5-fold increase in the mutant frequency when AG30 was pre-incubated with the supFG1 plasmid in the absence of potassium prior to transfection into COS-7 cells. However, when we analysed triplex formation of AG30 and the supFG1 target duplex at physiological potassium levels, triplex formation was inhibited due to the formation of competing secondary structures. Subsequent assessment of mutant frequency under physiological conditions, by pre-transfecting COS-7 cells with the supFG1 plasmid prior to AG30 treatment led to a very small increase (1.4-fold) in the mutant frequency. Transfection of cells with even higher concentrations of AG30 did result in an elevated mutagenic response but this was also seen with a scrambled sequence, and was therefore considered unlikely to be biologically relevant as an associated increase in cytotoxicity was also apparent. Our findings also provide further assurance on the low potential of triplex-mediated mutation as a consequence of unintentional genomic DNA binding by therapeutic antisense oligonucleotides.
Issue Date: 21-Sep-2016
Date of Acceptance: 8-Sep-2016
URI: http://hdl.handle.net/10044/1/41184
DOI: https://dx.doi.org/10.1093/toxsci/kfw179
ISSN: 1096-6080
Publisher: Oxford University Press
Start Page: 101
End Page: 111
Journal / Book Title: Toxicological Sciences
Volume: 155
Issue: 1
Copyright Statement: © 2016 Oxford University Press. This is a pre-copy-editing, author-produced PDF of an article accepted for publication in Toxicological Sciences following peer review. The definitive publisher-authenticated version is available online at: http://dx.doi.org/10.1093/toxsci/kfw179.
Sponsor/Funder: British Heart Foundation
Imperial College Trust
THE LEVERHULME TRUST
Funder's Grant Number: PG/11/81/29130
N/A
RGP-2012-603
Keywords: antisense oligonucleotides
mutagenesis.
supF assay
triplex forming oligonucleotides
Toxicology
1115 Pharmacology And Pharmaceutical Sciences
Publication Status: Published
Appears in Collections:Division of Surgery
National Heart and Lung Institute
Faculty of Medicine



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