Triplex forming oligonucleotides (TFOs) are some of the most promising tools in the antigene strategy for the development of gene targeting therapeutics. However, the stable triplex formation is restricted to the homopurine sequences consisting of purine nucleosides, dG and dA. Therefore, the T or dC nucleoside in the homopurine strand inhibits the stable triplex formation. We have developed W-shaped nucleoside analogues (WNAs) for the formation of the unnatural type triplex DNA, with sequences containing the interrupting site in an antiparallel triplex formation. In the present study, we tested the antigene effect of TFOs having WNA-βT, which increased the stability of the triplex formation with a target sequence including the TA interrupting site. We designed the GU TFO (WNA) and GU TFO (natural) for targeting sequences of the Bcl-2 or survivin oncogene. The gel shift assay showed that the TFO (WNA) formed more stable triplexes than the natural TFO. Remarkably, the Bcl-2- or survivin-targeted TFO (WNA) inhibited the cell proliferation and induced a caspase-dependent apoptosis. It was confirmed that the survivin-targeted TFO (WNA) more effectively decreased the number of survivin products in the A549 cell than the natural TFOs.

The antigene strategy based on site-specific recognition of duplex DNA by triplex DNA formation has been exploited in a wide range of biological activities. However, specific triplex formation is mostly restricted to homo-purine strands within the target duplex DNA, due to the destabilizing effect of CG and TA inversion sites where there is an absence of natural nucleotides that can recognize the CG and TA base pairs. Hence, the design of artificial nucleosides, which can selectively recognize these inversion sites with high affinity, should be of great significance. Recently, we determined that 2-amino-3-methylpyridinyl pseudo-dC (^3MeAP-ΨdC) possessed significant affinity and selectivity toward a CG inversion site and showed effective inhibition of gene expression. We now describe the design and synthesis of new modified aminopyridine derivatives by focusing on small chemical modification of the aminopyridine unit to tune and enhance the selectivity and affinity toward CG inversion sites. Remarkably, we have newly found that 2-amino-4-methoxypyridinyl pseudo-dC (^4OMeAP-ΨdC) could selectively recognize the CG base pair in all four adjacent base pairs and form a stable triplex structure against the promoter sequence of the human gene including multiple CG inversion sites.

Expansion of the triplex DNA forming sequence is required in the genomic targeting fields. Basically, triplex DNA is formed by the interaction between the triplex-forming oligonucleotides and homo-purine region with the target duplex DNA. The presence of the base pair conversion sites hampers stable triplex formation. To overcome this limitation, it is necessary to develop an artificial nucleic acid to recognize the base conversion sites, and the CG and TA base pairs. We describe the synthesis of C-nucleoside analogues and an evaluation of the ability of triplex formation. Consequently, the combined use of the novel C-nucleoside analogues, AY – AY-d(Y-NH₂), AY-d(Y-Cl) and ^IAP-d(Y-Cl), is capable of recognizing duplex DNA including the TA or dUA base pair.

The development of novel nucleoside analogues for the formation of triplex DNA containing pyrimidine–purine inversion sites has been a challenging field. In this paper, we describe the design and synthesis of non-natural nucleoside analogues, N-substituted-2′-deoxy-5-methylisocytidine derivatives, and their evaluation for triplex formation. It has been shown that N-(guanidinoethyl)-2′-deoxy-5-methylisocytidine exhibits selective recognition of a CG interrupting site and potentiates the formation of anti-parallel triplexes.