Park W S, Miyano-Kurosaki N, Abe T, Takai K, Yamamoto N, Takaku H
Department of Industrial Chemistry, High Technology Research Center, Chiba Institute of Technology, 2-17-1 Tsudanuma, Narashino, Chiba, 275-0016, Japan.
Biochem Biophys Res Commun. 2000 Apr 21;270(3):953-60. doi: 10.1006/bbrc.2000.2542.
We have designed a new class of oligonucleotides, "dumbbell RNA/DNA chimeric phosphodiesters," containing two alkyl loop structures with RNA/DNA base pairs [sense (RNA) and antisense (DNA)] in the double helical stem. The reaction of nicked (NDRDON-gag-AUG) and circular (CDRDON-gag-AUG) dumbbell RNA/DNA chimeric oligonucleotides with RNaseH gave the corresponding antisense phosphodiester oligonucleotide together with the sense RNA cleavage products. The liberated antisense phosphodiester oligodeoxynucleotide was bound to the target RNA. The circular dumbbell RNA/DNA chimeric oligoncleotide showed more nuclease resistance and cellular uptake than the linear antisense phosphorothioate oligodeoxynucleotide (S-ODN-gag-AUG) and nicked dumbbell RNA/DNA chimeric oligonucleotide. The CDRDON-gag-AUG with an AUG initiation codon sequence, as the target of the HIV-1 gag-gene (779-801), was synthesized and tested for inhibitory effects using peripheral blood mononuclear cells. The circular dumbbell RNA/DNA chimeric oligonucleotide (CDRDON-gag-AUG) showed highly inhibitory effects compared to the antisense phosphorothioate oligonucleotide (S-ODN-gag-AUG), indicating sequence-specific inhibition of HIV-1 replication without the inhibition of reverse transcriptase and/or the viral entry process such as antisense phosphorothioate oligonucleotides.
我们设计了一类新型的寡核苷酸，即“哑铃状RNA/DNA嵌合磷酸二酯”，其在双螺旋茎中包含两个带有RNA/DNA碱基对[正义链（RNA）和反义链（DNA）]的烷基环结构。带切口的（NDRDON-gag-AUG）和环状的（CDRDON-gag-AUG）哑铃状RNA/DNA嵌合寡核苷酸与核糖核酸酶H反应，产生了相应的反义磷酸二酯寡核苷酸以及正义链RNA裂解产物。释放出的反义磷酸二酯寡脱氧核苷酸与靶RNA结合。与线性反义硫代磷酸酯寡脱氧核苷酸（S-ODN-gag-AUG）和带切口的哑铃状RNA/DNA嵌合寡核苷酸相比，环状哑铃状RNA/DNA嵌合寡核苷酸表现出更强的核酸酶抗性和细胞摄取能力。合成了带有AUG起始密码子序列的CDRDON-gag-AUG，其作为HIV-1 gag基因（779-801）的靶标，并使用外周血单核细胞测试其抑制作用。与反义硫代磷酸酯寡核苷酸（S-ODN-gag-AUG）相比，环状哑铃状RNA/DNA嵌合寡核苷酸（CDRDON-gag-AUG）显示出高度的抑制作用，表明其对HIV-1复制具有序列特异性抑制作用，而不像反义硫代磷酸酯寡核苷酸那样抑制逆转录酶和/或病毒进入过程。
