Unambiguous demonstration of triple-helix-directed gene modification

Abstract

Triple-helix-forming oligonucleotides (TFOs), which can potentially modify target genes irreversibly, represent promising tools for antiviral therapies. However, their effectiveness on endogenous genes has yet to be unambiguously demonstrated. To monitor endogenous gene modification by TFOs in a yeast model, we inactivated an auxotrophic marker gene by inserting target sequences of interest into its coding region. The genetically engineered yeast cells then were treated with psoralen-linked TFOs followed by UV irradiation, thus generating highly mutagenic covalent crosslinks at the target site whose repair could restore gene function; the number of revertants and spectrum of mutations generated were quantified. Results showed that a phosphoramidate TFO indeed reaches its target sequence, forms crosslinks, and generates mutations at the expected site via a triplex-mediated mechanism: (i) under identical conditions, no mutations were generated by the same TFO at two other loci in the target strain, nor in an isogenic control strain carrying a modified target sequence incapable of supporting triple-helix formation; (ii) for a given target sequence, whether the triplex was formed in vivo on an endogenous gene or in vitro on an exogenous plasmid, the nature of the mutations generated was identical, and consistent with the repair of a psoralen crosslink at the target site. Although the mutation efficiency was probably too low for therapeutic applications, our results confirm the validity of the triple-helix approach and provide a means of evaluating the effectiveness of new chemically modified TFOs and analogs.

Figure 1

Analysis of TFOs in vitro and on exogenous plasmids. (A) Sequences of the target gene and the TFO (shown bound on the target gene); two variants of the TFO were used with identical length and sequence but differing in their chemical modification (see text). The ochre codon is indicated in bold. The cytosine in the oligonucleotide is methylated. The furane (F) and pyrone (P) sides of the adduct are indicated, as is the position of the DraI restriction site. (B) The PPN TFO binds to the target sequence with greater affinity than does the PPO TFO. A plasmid including the target sequence (YEplac181_ura3∷hiv1pur) was incubated with increasing doses of TFOs, irradiated with near UV light, and submitted to DraI restriction. Shown are dose curves of DraI inhibition by the PPN or the PPO oligonucleotides, as indicated. (C) Frequency of the mutations generated on an exogenous episomal plasmid (YEplac112_ura3∷hiv1pyr) by the PPO TFO, the PPN TFO, or in the absence of TFO (UVA). Triple helices were preformed on plasmids in vitro. Samples were irradiated, resulting in the introduction of covalent crosslinks into a high proportion of the target plasmids—proportions, which, under the conditions used, were similar for both TFOs as shown by renaturing gel electrophoresis analysis (Left; DS, double-stranded, crosslinked species; SS, single-stranded, noncrosslinked species). The plasmids were used to transform ura3–52 yeast cells. The mutation frequency was defined as the ratio between the number of induced URA3+ revertants and the total number of transformants. Shown is the mean value of three independent experiments.

Figure 2

A PPN TFO generates mutations in a cognate endogenous target sequence and not in unrelated genes. Number of revertants generated by a PPO TFO, a PPN TFO, or in the absence of TFO (UVA), as indicated, on the HIV-1 target sequence inserted into the endogenous URA3 gene (ura3∷hiv-1), or on irrelevant genes (ade2–101 and lys2–801) of a CmY826-derived strain. TFOs were electroporated into intact yeast cells, cells were irradiated, and URA3+, ADE2+, or LYS2+ revertants were selected on the appropriate medium. Shown is the mean value of three independent experiments. The HIV-1 target sequence is shown in Fig. 4A.

Figure 3

The frequency and types of mutations generated by the PPN TFO depend on the orientation of the target sequence with respect to the gene promoter. (A) Sequence of the target in the direct and inverted orientations showing the preferential orientation of the psoralen crosslink positioned by the TFO. (B) Number of revertants generated at the target site in the direct and inverted orientations. CmY826-derived cells carrying a direct or inverted HIV-1 PPT were electroporated and selected as in Fig. 2. (C) Mutations generated on exogenous or endogenous targets are similar in nature. Graphs indicate the proportion of each type of mutation, determined by sequencing the mutants obtained through UVA irradiation (UVA on chromosome), through action of the PPN TFO on endogenous HIV-1 PPT targets (PPN on chromosome), or in experiments in which the triplex was preformed on plasmids (YEplac112_ura3∷hiv1pur and _ura3∷hiv1pyr) in vitro (PPN on plasmid). The number of mutants analyzed is indicated below each graph. A similar effect of the orientation of the target was observed in the FF18 733 background, so that sequencing results obtained in both the CmY826 and the FF18 733 backgrounds were pooled.

Figure 4

Cross-reaction between HIV-1 and HIV-2 oligopurine sequences. (A) Sequences of the HIV-1, HIV-2, and control (Cont) targets. The triple-helix target is boxed, and the nucleotides that differ from the HIV-1 sequence are indicated by darker boxes in the HIV-2 and control target sequences. (B) Association of PPN TFO with HIV-1 and HIV-2 in vitro. Dose curves of DraI restriction enzyme inhibition by the PPN oligonucleotide on plasmids including the HIV-1 or the HIV-2 target (YEplac181_ura3∷hiv1pur and _ura3∷hiv2pur), as indicated. (C) Number of revertants generated at the target site by the PPN TFO on HIV-1, HIV-2, or control endogenous targets, as indicated. FF18 733-derived cells harboring the HIV-1, HIV-2, or control sequence shown in Fig. 4A were electroporated, irradiated, and selected as in Fig. 2.