Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
Graduate School of Pharmaceutical Sciences, Nagasaki International University, 2825-7 Huis Ten Bosch Machi, Sasebo city, Nagasaki 859-3298, Japan.
Nucleic Acids Res. 2022 Nov 28;50(21):12071-12081. doi: 10.1093/nar/gkac1110.
The formation of triplex DNA is a site-specific recognition method that directly targets duplex DNA. However, triplex DNA formation is generally formed for the GC and AT base pairs of duplex DNA, and there are no natural nucleotides that recognize the CG and TA base pairs, or even the 5-methyl-CG (5mCG) base pair. Moreover, duplex DNA, including 5mCG base pairs, epigenetically regulates gene expression in vivo, and thus targeting strategies are of biological importance. Therefore, the development of triplex-forming oligonucleotides (TFOs) with artificial nucleosides that selectively recognize these base pairs with high affinity is needed. We recently reported that 2'-deoxy-2-aminonebularine derivatives exhibited the ability to recognize 5mCG and CG base pairs in triplex formation; however, this ability was dependent on sequences. Therefore, we designed and synthesized new nucleoside derivatives based on the 2'-deoxy-nebularine (dN) skeleton to shorten the linker length connecting to the hydrogen-bonding unit in formation of the antiparallel motif triplex. We successfully demonstrated that TFOs with 2-guanidinoethyl-2'-deoxynebularine (guanidino-dN) recognized 5mCG and CG base pairs with very high affinity in all four DNA sequences with different adjacent nucleobases of guanidino-dN as well as in the promoter sequences of human genes containing 5mCG base pairs with a high DNA methylation frequency.
三链 DNA 的形成是一种针对双链 DNA 的特异性识别方法。然而，三链 DNA 的形成通常针对双链 DNA 的 GC 和 AT 碱基对形成，并且没有天然核苷酸可以识别 CG 和 TA 碱基对，甚至是 5-甲基-CG（5mCG）碱基对。此外，包括 5mCG 碱基对在内的双链 DNA 在体内通过表观遗传调控基因表达，因此靶向策略具有生物学重要性。因此，需要开发具有人工核苷的能够高亲和力选择性识别这些碱基对的三链形成寡核苷酸（TFO）。我们最近报道了 2'-脱氧-2-氨基内消旋-胞苷衍生物具有在三链形成中识别 5mCG 和 CG 碱基对的能力；然而，这种能力取决于序列。因此，我们基于 2'-脱氧-内消旋胞苷（dN）骨架设计并合成了新的核苷衍生物，以缩短形成反平行基序三链时连接到氢键单元的连接子长度。我们成功地证明了具有 2-胍基乙基-2'-脱氧内消旋胞苷（胍基-dN）的 TFO 能够以非常高的亲和力识别所有四个具有不同相邻碱基的 dN 以及含有高 DNA 甲基化频率的 5mCG 碱基对的人类基因启动子序列中的 5mCG 和 CG 碱基对。
