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

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 pretransfecting 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.

An Oligo-Library-Based Approach for Mapping DNA–DNA Triplex Interactions In Vitro

“…We carried out the Triplex-seq experiments in two different buffers: pH 7, and triplex-disfavoring high potassium pH 7, with a TTS (TTS-1; see Table ) that was previously shown using EMSA to form a triplex with a specific TFO. High potassium buffers are triplex-disfavoring, and instead have been shown to stabilize G-quadruplex structures. − After sequencing, we counted the number of normalized reads that were attained for each variant for both the triplex and TFO-only control lane and plotted them in Figure C (left).…”

“…Here the “-” denotes Watson–Crick base-pairing between the purine base R and its pyrimidine pair Y, and “*” denotes Hoogstein interaction. TFOs have subsequently been used to identify triplex rules , and have been utilized as biotechnological tools in vitro and in vivo . − …”

Identifying triplex binding rulesin vitroleads to creation of a new synthetic regulatory toolin vivo

“…We next sought to evaluate which TFO sequences will be enriched when testing the ~ 270,000,000 large N-TFO library (Figure 3a, top). We used two known TTSs (pH 5: TTS 2) 41 and (pH 7: TTS 1) 24 as our test targets ( Figure 3a, middle and bottom) alongside the N-TFO library to determine the migration pattern of duplexes and triplexes in various buffer conditions. In Figure 3b, we plot the mean nucleotide frequency of the enriched TFO sequences against the triplex reactivities for pH 5 (Figure 3b, left) and pH 7 (Figure 3b, right) and observe a G-increase in the TFO sequences with higher triplex reactivities for both conditions.…”

“…In Figure 3b, we plot the mean nucleotide frequency of the enriched TFO sequences against the triplex reactivities for pH 5 (Figure 3b, left) and pH 7 (Figure 3b, right) and observe a G-increase in the TFO sequences with higher triplex reactivities for both conditions. To further characterize the behavior of the N-TFO, we additionally applied the Triplex-Seq platform in a buffer that negatively affects triplex formation due to the presence of monovalent ions ( Figure 3c) 24,42 . In Figure 3d the triplex reactivities of both TTS sequences in the triplex-disfavoring buffer are plotted and we observe again a trend toward Grich sequences with higher triplex reactivities, but this effect is weaker as compared to the conditions lacking potassium ( Figure 3b).…”

“…Due to their specificity in targeting dsDNA, TFOs were subsequently used to identify triplex rules 13,14 and applied as biotechnological tools in vitro and in vivo to regulate transcription [15][16][17][18] , control recombination events 19 and induce triplex-directed mutagenesis [20][21][22] . Despite extensive amount of work to establish TFOs as therapeutic agents, triplex formation in vivo mostly was attenuated and the expected biological function was limited 23,24 .…”