Triplex-forming oligonucleotides (TFOs) are potential DNA-targeting molecules and would become powerful tools for genomic research. As the stabilization of the TFO is partially provided by hydrogen bonds to purine bases, the most stable triplexes form with homopurine/homopyrimidine sequences, and a pyrimidine base in the purine strand of the duplex interrupts triplex formation. If a TFO can recognize sequences including such an interrupting site, the target regions in the genome would be expanded to a greater extent. However, this problem has not been generally solved despite extensive studies. We have previously reported a new base analogue (WNA) constructed of three parts, a benzene ring, a heterocyclic ring, and a bicyclic skeleton to hold these two parts. In this study, we have further investigated modification of WNA systematically and determined two useful WNA analogues, WNA-beta T and WNA-beta C, for selective stabilization of triplexes at a TA and a CG interrupting site, respectively. The triplexes with WNA analogues have exhibited an interesting property in that they are more stable than natural-type triplexes even at low Mg(2+) concentration. From comparison of the results with H-WNA-beta T lacking benzene and those with WNA-H without thymine, it has been suggested that benzene is a major contributor for triplex stability and thymine provides selectivity. Thus, it has been successfully demonstrated that WNA-beta T/TA and WNA-beta C/CG combinations may expand triplex recognition codes in addition to the natural A/AT and G/GC base triplet codes. The results of this study will provide useful information for the design of new WNA analogues to overcome inherent problems for further expansion of triplex recognition codes.
Triplex-forming oligonucleotides (TFOs) are sequence-specific DNA-binding agents, but their target duplexes are limited to homopurine/homopyrimidine sequences because of interruption of the pyrimidines bases in the purine region. This problem has not been fully solved despite a wide variety of studies. Recently, we have developed a bicyclic system as a novel scaffold for nucleoside analogues (WNA, W-shaped nucleoside analogues) and determined two useful compounds, WNA-betaT (2) and WNA-betaC (5), for highly stable and selective triplex formation at a TA and a CG interrupting site, respectively. However, subsequent investigations have shown that the triplex formation using WNA is dependent on the neighboring bases of the TFOs. In this study, we have synthesized new WNA derivatives having halogenated recognition bases or benzene rings and evaluated the effects of the modifications on the triplex stability as well as selectivity. It has been found that the WNA-betaT analogues holding 5-halogenated pyrimidine bases (WNA-beta(Br)U (3) and WNA-beta(F)U (4)) exhibit high CG-selectivity. On the other hand, the WNA-betaT derivatives having the bromo-substituted benzene ring (mBr-WNA-betaT (10) and oBr-WNA-betaT (11)) have shown high selectivity to a TA interrupting site with high stability in the sequences to which the original WNA-betaT do not bind. Thus, sequence-dependency has been overcome by the sequence-dependent use of WNA-betaT, mBr-WNA-betaT, and oBr-WNA-betaT.
Recently, we have developed new base analogs (WNA) and demonstrated that WNA-[see text];T with thymine and WNA-[see text];C with cytosine stabilize n on-natural antiparallel triplexes with a TA or CG interrupting site, respectively. However, limitations in recognizable sequences with the WNA-containing TFO were also found. The objective of this study is to search better WNA analogs for expansion of triplex recognition codes to general duplex sequences. In this study, we designed new WNA analogs by systematic modification of the aromatic part and the recognition part. The new WNA analogs with the benzene ring substituted with bromide or cyanide have determined for selective stabilization of triplexes at a TA interrupting site, and general formation of triplexes having a TA interrupting site has been achieved.
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