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

新一代2-5A-反义蛋白的开发及其基因表达调控特性。

基本信息

批准号：
15390036
负责人：
KITADE Yukio
金额：
$ 8.58万
依托单位：
Gifu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15390036/
关键词：
2-5A antisense 2-5A-antisense ribonuclease oligomer messenger RNA

项目摘要

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.

1.8甲基腺苷取代的2-5A四聚体在5′-磷酸基团上具有羟基烷基，用磷酸酰胺法在DNA/RNA合成器上合成。在2-5A四聚体和2-5A反义嵌合体中加入羟乙基后，其类似物对重组人rna酶L的激活能力略有降低，但与不含羟乙基和8-甲基腺苷的寡核苷酸相比，含有羟乙基和8-甲基腺苷的2-5A四聚体和2-5A反义嵌合体的激活能力分别恢复到80%和50%。合成了一种新的2- 5a -反义嵌合体，其反义部分的5′端含有2-（羟甲基）-1,3-丙二醇连接体的2- 5a四聚体分子。双头2- 5a -反义嵌合体更有效地切割靶rna。我们检测了含有2'-脱氧-2'-α-氟尿嘧啶或2'- o -甲基尿嘧啶的RNA类似物作为抑制人RNA酶L的性质，该RNA酶L在2',5'-连接的低聚腺苷酸（2- 5a）存在下切割单链RNA。在检测的单氨基酸突变体中，Y712A和F716A导致RNase活性显著降低，RNA结合活性降低。结果发现，在5′端第2位和第3位含有一个无环核苷的类似物的效力仅比亲本2-5A四聚体低9倍和1.7倍。我们展示了人类RNase L的n端锚蛋白重复结构域与含有2‘,5’核苷酸间键的激活剂2- 5a络合的晶体结构。ANK识别2-5A的结构基础对于设计稳定的、高激活RNase L.7可能性的2-5A至关重要。RNase L的锚定重复结构域通过结合2- 5a - 8收缩其结构。靶向病毒mRNA的2- 5a -反义嵌合体的合成及其抗病毒活性正在研究中。

项目成果

期刊论文数量（27）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Synthesis of antisense oligonucleotides carrying modified 2-5A molecules at their 5'-termini and their properties.

5 末端携带修饰的 2-5A 分子的反义寡核苷酸的合成及其特性。

DOI：
发表时间：
2003
期刊：
Bioconjugate Chemistry 14
影响因子：
0
作者：
Sakanaka;M.;Hatae;N.;Tanaka;S.;Senma;M.;Ichikawa;A.;Keiko Kashiwagi et al.;Sasaki et al.;Nao Odagiri;上野義仁
通讯作者：
上野義仁

2-5A Induces a Conformational Change in the Ankyrin-repeat Domain of RNase L.

2-5A 诱导 RNase L 锚蛋白重复结构域的构象变化。

DOI：
发表时间：
2005
期刊：
PROTEINS (in press)
影响因子：
0
作者：
Arai;K. et al.;Seiki Hirano;Kotani et al.;中西雅之
通讯作者：
中西雅之

Interferon-a and antisense K-ras RNA combination gene therapy against pancreatic cancer

干扰素-a 和反义 K-ras RNA 组合基因治疗胰腺癌

DOI：
发表时间：
2004
期刊：
The Journal of Gene Medicine 6
影响因子：
0
作者：
Sugimoto;Y.;Tsuboi;H.;Okuno;Y.;Tamba;S;Tsuchiya;S.;Tsujimoto;C.;Ichikawa;A.;Hiroshi Yamazaki et al.;Daisuke Maeda;Kondo et al.;Kaori Takehara et al.;Kazuteru Hatanaka
通讯作者：
Kazuteru Hatanaka

Contribution of Tyr712 and Phe716 to the Activity of Human RNase L.

Tyr712 和 Phe716 对人 RNase L 活性的贡献。