A triple-helix forming oligonucleotide targeting genomic DNA fails to induce mutation

Abstract

Purine tracts in duplex DNA can bind oligonucleotide strands in a sequence specific manner to form triple-helix structures. Triple-helix forming oligonucleotides (TFOs) targeting supFG1 constructs have previously been shown to be mutagenic raising safety concerns for oligonucleotide-based pharmaceuticals. We have engineered a TFO, TFO27, to target the genomic Hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus to define the mutagenic potential of such structures at genomic DNA. We report that TFO27 was resistant to nuclease degradation and readily binds to its target motif in a cell free system. Contrary to previous studies using the supFG1 reporter construct, TFO27 failed to induce mutation within the genomic HPRT locus. We suggest that it is possible that previous reports of triplex-mediated mutation using the supFG1 reporter construct could be confounded by DNA quadruplex formation. Although the present study indicates that a TFO targeting a genomic locus lacks mutagenic activity, it is unclear if this finding can be generalised to all TFOs and their targets. For the present, we suggest that it is prudent to avoid large purine stretches in oligonucleotide pharmaceutical design to minimise concern regarding off-target genotoxicity.

The Lack of Mutagenic Potential of a Guanine-Rich Triplex Forming Oligonucleotide in Physiological Conditions

“…Consistent with these results, we previously observed that quadruplex formation by AG30 is stabilised by potassium and that replacing four guanine residues with adenines to interrupt the contiguous run of guanines within AG30 attenuates quadruplex formation (Reshat et al, 2012). Similar results have recently been reported where partial substitution of AG30 with 8-aza-7-deaza-guanine can improve its bioactivity (Rogers et al, 2014).…”

“…However, it should be noted that co-localisation was assessed 4 hours after transfection while cells incubated with the oligonucleotide were assessed for mutagenesis over two days, it is possible that with additional time more of the oligonucleotide would co-localise with the plasmid DNA. In fact, the mutagenic activity induced by AG30, after Oligofectamine transfection into cells, suggests a fraction of the (Cheng and Van Dyke, 1997;Reshat et al, 2012;Rogers et al, 2014). Indeed, in the presence of physiological potassium concentration, we could not detect triplex formation using gel mobility analysis even at the highest concentration of AG30 used.…”

“…Finally, as mentioned above, triplex formation could be influenced by the accessibility of the target gene in the genomic DNA and the stability of triplexes under physiological ionic conditions (Macris and Glazer, 2003;Lin et al, 2000). In our earlier study, a 27mer TFO (TFO27) designed to bind to a purine rich tract in the HPRT locus, was unable to induce mutation at the chromosomal targetin human lymphoblastoid TK6 cells even though this TFO was able to bind to its target with high affinity in a cell free system (Reshat et al, 2012). Therefore, while the theoretical potential for triplex target sequences could raise a mutagenic concern; it does not guarantee the bioactivity of TFOs as it could vary from one gene target to another depending on their transcription levels and accessibility to TFO as mentioned above (Macris and Glazer 2003).…”