Design of the new recognition molecules for the formation of triplex helix DNA at any predetermined sites.

自从双链DNA主槽内的三链形成被认为是一种选择性地抑制预定序列上的基因表达的方法以来，人们主要关注的是克服其固有的局限性，即三链仅针对双链的同嘌呤-同嘧啶序列形成。也就是说，双链高嘌呤链内的嘧啶碱基抑制三链的形成；因此，人们一直致力于开发非天然碱基结构来稳定这些中断位点上的三链。然而，在任何DNA序列上形成三链仍然是一个具有挑战性的课题。在这种方法中，我们专注于由富含嘌呤的三链形成寡核苷酸(TFO)形成的反平行三链，因为反平行三链在生理条件下形成的稳定性比含有嘧啶TFOs的反平行三链形成的稳定性更高。我们设计了一个新的核苷类似物的一般结构，它含有用于堆积的芳香部分，具有Hoogesen氢键的碱基，以及一个[3.3.0]双正辛烷结构来固定前两个组分。新设计的分子因其形状而被命名为W形核酸(WNA)。在反平行方向上，嘌呤碱基有望在双链的嘧啶链中形成两个指向目标嘌呤碱基的Hoogsteen氢键，芳香族可能通过新的核苷类似物起到维持TFO堆积作用的作用。综上所述，我们揭示了新的W型核酸衍生物WNA-BT对TA中断位点具有高选择性的稳定作用。此外，WNA-BC显示出对具有CG中断位点的双链的选择性稳定。因此，新的WNA类似物将成为形成对TA和CG中断位点具有高选择性和亲和力的非自然型三联体的新候选者。

Since triplex formation within the major groove of duplex DNA has been proposed as a selective method for specific inhibition of gene expression at a predetermined sequence, a major concern has been to overcome its intrinsic limitation that triplexes are formed only toward homopurine-homopyrimidine sequences of the duplex. That is, pyrimidine bases within the homopurine strand of the duplex inhibit triplex formation; therefore, efforts have been focused on development of a non-natural base structure to stabilize triplexes at such interrupting sites. Nevertheless, triplex formation at any DNA sequence has remained a challenging theme.In this approach, we focused on an antiparallel triplex formed with purine-rich triplex-forming oligonucleotides (TFO), because the antiparallel triplexes are formed under physiological conditions with higher stability than the parallel ones with pyrimidine TFOs.We designed a new general structure of nucleoside analogs bearing an aromatic part for stacking, a base for Hoogesteen hydrogen bonds, and a [3.3.0]bicycooctane structure to fix fix the two former components. The newly designed molecule was named the W-shaped nucleic acid (WNA) after its shape. In the antiparallel orientation, the purine base is expected to form two Hoogesteen hydrogen bonds toward the target purine base within a pyrimidine strand of the duplex, and an aromatic may play a role to maintain stacking interaction of the TFO continuously through the new nucleoside analog.In conclusion, we have revealed that the new W-shaped nucleic acid derivative WNA-bT exhibits high stabilization effect toward a TA interrupting site with high selectivity. In addition, WNA-bC showed selective stabilization toward the duplex having a CG interrupting site. Thus, the new WNA analogs would become new candidates for the formation of non-natural type triplexes with high selectivity and affinity to a TA and to a CG interrupting site.