Development of a new generation of 2-5A-antisense and its property for the regulation of gene expression.

用DNA/RNA合成仪通过亚磷酰胺法合成了在5 ′-磷酸酯上具有羟烷基的1.8-甲基腺苷取代的2-5A四聚体和相应的嵌合体。将羟乙基并入2-5A四聚体和2- 5A-反义嵌合体中略微降低了它们的类似物激活重组人RNA酶L的能力，但是具有羟乙基和8-甲基腺苷的2-5A四聚体和2- 5A-反义嵌合体的能力相对于不具有羟乙基和8-甲基腺苷的寡核苷酸恢复到80%和50%，2.合成了一种新的2- 5A-反义嵌合体，其在反义部分的5 ′-末端具有两个分子的2-5A四聚体，并具有2-（羟甲基）-1，3-丙二醇接头。2- 5A反义双头嵌合体能更有效地切割靶RNA。3.我们检测了含有2 ′-脱氧-2 ′-α-氟尿苷或2 ′-O-甲基尿苷的RNA类似物作为人RNase L抑制剂的性质，这些RNA类似物在2 ′，5 ′-连接的寡腺苷酸（2-5A）存在下切割单链RNA。Y 712 A和F716 A导致RNase活性显着降低，RNA结合活性降低。5.发现在5 '端第二位和第三位含有无环核苷的类似物的效力仅比母体2-5A四聚体低9倍和1.7倍。6.我们展示了N-四聚体的晶体结构与含有2 '，5'核苷酸间键的激活剂2-5A复合的人RNA酶L的末端锚蛋白重复结构域。ANK识别2-5A的结构基础是设计稳定的2- 5A并使其具有较高的激活RNase L的可能性的必要条件。7. RNase L的锚定重复结构域通过与2- 5A结合而限制其结构。8.针对病毒mRNA的2- 5A反义嵌合体的合成及其抗病毒活性研究正在进行中。

1.8-Methyladenosine-substituted 2-5A tetramers with hydroxyalkyl groups at the 5'-phosphates and the corresponding chimeras were synthesized by the phosphoramidite method with a DNA/RNA synthesizer. Incorporation of the hydroxyethyl group into 2-5A tetramer and 2-5A-antisense chimera slightly reduced the abilities of their analogs to activate recombinant human RNase L, but the abilities of the 2-5A tetramer and the 2-5A-antisense chimera both with hydroxyethyl group and 8-methyladenosine returned to 80 and 50% relative to those of oligonucleotides without the hydroxyethyl group and 8-methyladenosine, respectively.2.A novel 2-5A-antisense chimera having two molecules of a 2-5A tetramer at the 5'-terminal of the antisense moiety with a 2-(hydroxymethyl)-1,3-propanediol linker was synthesized. The double-headed 2-5A-antisense chimeras more efficiently cleaved the target RNA.3.We examined the properties of RNA analogs containing 2'-deoxy-2'-α-fluorouridine or 2'-O-methyluridine as inhibitors against human RNase L, that cleaved a single-stranded RNA in the presence of 2',5'-linked oligoadenylate (2-5A).4.Among the single amino acid mutants examined, Y712A and F716A resulted in a significant decrease of RNase activity with a reduced RNA binding activity.5.It was found the analogs containing an acyclonucleoside at the second position and at the third position from the 5'-end were only 9-and 1.7-fold less potent than the parent 2-5A tetramer.6.We presented the crystal structure of the N-terminal ankyrin repeat domain of human RNase L complexed with activator 2-5A containing 2',5' internucleotide linkages. The structure basis for 2-5A recognition by ANK was essential for designing stable 2-5As with high likelihood of activating RNase L.7.The ankyrin-repeat domain of RNase L constricted its structure by binding of 2-5A.8.The synthesis of 2-5A-antisense chimera targeting a viral mRNA and its antiviral activity are now in progress.