Development and clinical trials of oligonucleotide therapeutics, including antisense oligonucleotide, siRNA, aptamer and decoy, are progressing around the world, but most of them target single-stranded RNA. However as the complexity of RNA regulatory mechanisms has been revealed in recent years, we have targeted double-stranded DNA (duplex DNA), which is the foundation of RNA control in the future The triplex DNA formation strategy, in which duplex DNA is formed by the interaction with the other single-stranded oligonucleotide, is a technique that can directly act on abnormal genes involved in disease and control the transcription step. This method is called the antigene method, but at present it has hardly been studied due to the intrinsic limitation on its target sequence. One of the problems is the presence of a mismatch site where the formation of the triplex DNA is interrupted. The other strand, dG or dA, interacts with the dG or dA, which is the purine base of the GC or AT base pair of target duplex DNA, respectively, to form the stable triplex DNA. For this reason, natural antigene oligonucleotides cannot recognize pyrimidine bases such as CG and TA base pairs in which the base pairs in duplex DNA have been exchanged, and cannot form the stable triplex DNA. On the other hand, when the target sequence is determined with the aim of forming stable and highly selective triplex DNA, the target sequence should contain a mismatch site, such as CG or TA base pairs, to suppress the off-target effect. Since it must be incorporated in at least one site, natural type of antigene oligonucleotides cannot be formed the triplex DNA. We have been developing artificial nucleic acids to selectively recognize CG or TA base pair. Recently, we achieved the development of novel nucleoside analogue capable of forming the stable non-natural type triplex DNA, and also succeeded in inhibition of the gene expression.
In this project, in order to position the antigene oligonucleotides having the artificial nucleoside analogues as one of the oligonucleotide therapeutics, we will use our original artificial nucleic acid as a basic technology. Then, we design and synthesize the antigene oligonucleotides with the artificial nucleoside analogues or chemical modification. By the clarifying the properties of these oligonucleotides, we will expand this development technology as new modalities for the drug discovery.
Figure 1: Structures of Base TripletsNatural base triplet, and Non-natural base triplet using artificial nucleic acids for base pair (CG or TA base pair) that cannot be stabilized by natural nucleoside.
Figure 2: Project AimsIn this project, we will synthesize an antigene oligonucleotide having an artificial nucleic acid. Cultured cells are treated with this antigene oligonucleotide to test the inhibitory effect of the mRNA transcription. (For example, the hTERT gene in this figure). Furthermore, we will clarify the transcription inhibitory activity targeting various genes.
Figure 3: DNA automatic synthesizer