Targeting of an interrupted polypurine:polypyrimidine sequence in mammalian cells by a triplex-forming oligonucleotide containing a novel base analogue

Abstract

The DNA triple helix consists of a third strand of nucleic acid lying in the major groove of an intact DNA duplex. The most stable triplexes form on polypurine:polypyrimidine sequences, and pyrimidine interruptions in the purine strand are destabilizing. Sequence stringency is imparted by specific Hoogsteen hydrogen bonds between third strand bases and the purine bases in the duplex. Appropriate base and sugar modifications of triple helix-forming oligonucleotides (TFOs) confer chromosome targeting activity in living cells. However, broad utilization of TFOs as gene targeting reagents in mammalian cells has been limited by the requirement for homopurine target sequences. Although there have been a number of base analogues described that appear to be promising as candidates for triplex target expansion, none has been examined in a biological system. We have employed a postsynthetic strategy to prepare a collection of TFOs with base analogues at a defined position. Following assessment of affinity for a triplex target with a single C:G inversion, TFOs with a second generation of analogues were synthesized. One of these, TFO-5a, with 2'-OMe-guanidinylethyl-5-methylcytosine at the position corresponding to the C:G interruption in the target sequence, was further modified to confer bioactivity. The activity of this TFO, linked to psoralen, was measured in a mammalian cell line that was engineered by directed sequence conversion to carry a triplex target with a single C:G interruption. TFO-5a was active against this target and inactive against the corresponding target with an uninterrupted polypurine:polypyrimidine sequence.

FIGURE 1

Schematic of base triplets formed in either an uninterrupted polypurine:polypyrimidine sequence or one with an “inverted” interruption in the sequence: (a) canonical G:C·C⁺, (b) inverted C:G·C, and (c) inverted C:G·C^mod. The R group is designed to afford additional hydrogen bonding between the base analogue and the purine base of the inverted base in the duplex target sequence.

FIGURE 2

Chinese hamster Hprt gene and psoralen triplex target sequence. The position that varies between mouse and hamster genomes is shown (X:Y) as is the location of base analogues in the third strand (Z). Mutagenesis by a TFO-targeted psoralen cross-link (that links a thymine on each duplex strand) results in small deletions in the target sequence and adjacent exon 5. The frequency of mutations can be determined by quantitation of thioguanine resistant colonies.

FIGURE 3

Analysis of base analogues in TFOs containing 2′-OMe ribose. (a) Bases and analogues in TFOs: 1, 2′-OMe thymidine; 2, 2′-OMe 5Me cytosine. (b) Binding assay of analogues shown in panel a. Resin-bound TFOs with base analogues were incubated with hairpin duplexes containing each of four possible base pairs at the X:Y position shown in Figure 2. After being washed, the remaining bound duplex was eluted from the resin (Methods) and the amount measured spectrophotometrically. (c) Thermal stability of 2′-OMe TFOs containing T (against the wild-type target sequence) or analogue 3c in the Z position.

FIGURE 4

Analysis of second generation analogues. (a) Analogue structure. (b) Binding assay as in Figure 3b. (c) Thermal stability of triplexes with 2′-OMe third strands. (d) Thermal stability profile of AE-TFO-5a on the duplex target with A:T or C:G at the X:Y position. The thick arrow denotes the inflection for the triplex, while the thin arrow indicates that for the duplex. There is a single transition in the pattern with the C:G duplex as the triplex is more stable than the underlying duplex. (e) Thermal stability of triplexes with the 2′-AE cluster.

FIGURE 5

Bioassay of TFO with the 5a analogue. (a) Construction of the CHO cell line with the murine triplex target site adjacent to exon 5 of Hprt. A cell line with a variant triplex target sequence adjacent to exon 5 was constructed by targeted sequence conversion (55, 56). This cell line is isogenic to the CHO AA8 line, differing only in the conversion of an A:T duplex to a C:G duplex at the X:Y position as shown in Figure 2. (b) Frequency of Hprt deficient colonies after treatment with AE-TFO-T and AE-TFO-5a in cells with the wild-type (A:T) and triplex (C:G) target.

FIGURE 6

Modeled structures of the central triplets formed by (A) A:T·T, (B) A:T·5MeC-5a, (C) G:C·T, (D) G:C·5MeC-5a, (E) C:G·T, and (F) C:G·5MeC-5a. The structures are snapshots from the 15 ns time step of the MD simulations and are oriented such that the viewer is looking down along the helical axis. In all systems, the base triplets are nearly coplanar. The strand 1 base is shown in standard CPK format, the strand 2 base in thin CPK format, and the strand 3 base in licorice format.

Scheme 1

Postsynthetic Solid Phase Synthesis of Base Analogues