针对人白细胞介素10基因产物的高效硫代磷酸酯反义寡核苷酸的设计及其在U937细胞中的反义活性评估。

Design of potent phosphorothioate antisense oligonucleotides directed to human interleukin 10 gene product and their evaluation of antisense activity in U937 cells.

作者信息

Arima H, Takahashi M, Aramaki Y, Sakamoto T, Yuda K, Akiyama K, Goto T, Tsuchiya S

机构信息

School of Pharmacy, Tokyo University of Pharmacy and Life Science, Hachioji, Japan.

出版信息

Pharm Res. 1999 Aug;16(8):1163-71. doi: 10.1023/a:1018964625977.

DOI:10.1023/a:1018964625977

PMID:10468015

Abstract

PURPOSE

The two objectives of this study were to design potent phosphorothioate antisense oligonucleotides (AS-S-oligos) directed against the human interleukin-10 (IL-10) gene product and to reveal the DNA sequence which best activates antisense effects.

METHODS

The design of potent AS-S-oligo was performed by using melting temperature (Tm) value of a DNA/RNA hybrid calculated by the nearest neighbor method and a secondary structure of human IL-10 mRNA suggested by RNA folding algorithms. U937 cells were used to estimate the antisense effect of the AS-S-oligos.

RESULTS

Of the eight candidates selected as potent AS-S-oligos on the basis of having higher Tm values and favorable secondary structures of the IL-10 mRNA, AS-S-oligos directed against the translated (AS367-S-oligo) and 3'-untranslated (AS637-S-oligo) region of IL-10 mRNA showed the strongest inhibitory effects on IL-10 production and this inhibition was dose- and time-dependent. Reverse transcription-polymerase chain reaction (RT-PCR) revealed that the antisense effects of AS-S-oligos originated from a specific reduction of target IL-10 mRNA by hybridization with AS367- and AS637-S-oligos. In addition, these AS-S-oligos did not affect human tumor necrosis factor-alpha (TNF-alpha) production in the cells stimulated by lipopolysaccharide (LPS). Strong positive correlations between the inhibitory effect of AS-S-oligos on the IL-10 production and not only Tm values calculated by nearest neighbor method but also Tm values determined by absorbance versus temperature profiles were demonstrated except for AS25-S-oligo and AS1249-S-oligo.

CONCLUSIONS

These findings suggest AS367- and AS637-S-oligos powerfully inhibit IL-10 production in U937 cells via an antisense mechanism. In addition, it is suggested efficiency of AS-S-oligo directed against the sequence of the target gene product can be explained by these Tm values and the proposed secondary structures of the target gene product.

摘要

目的

本研究的两个目标是设计针对人白细胞介素10（IL-10）基因产物的高效硫代磷酸酯反义寡核苷酸（AS-S-寡核苷酸），并揭示最能激活反义效应的DNA序列。

方法

通过使用最近邻法计算的DNA/RNA杂交熔解温度（Tm）值以及RNA折叠算法推测的人IL-10 mRNA二级结构来设计高效AS-S-寡核苷酸。使用U937细胞评估AS-S-寡核苷酸的反义效应。

结果

基于具有较高的Tm值和IL-10 mRNA的有利二级结构而被选为高效AS-S-寡核苷酸的八个候选物中，针对IL-10 mRNA翻译区（AS367-S-寡核苷酸）和3'-非翻译区（AS637-S-寡核苷酸）的AS-S-寡核苷酸对IL-10产生显示出最强的抑制作用，并且这种抑制是剂量和时间依赖性的。逆转录-聚合酶链反应（RT-PCR）显示，AS-S-寡核苷酸的反义效应源于与AS367-和AS637-S-寡核苷酸杂交导致靶IL-10 mRNA的特异性减少。此外，这些AS-S-寡核苷酸不影响脂多糖（LPS）刺激的细胞中人肿瘤坏死因子-α（TNF-α）的产生。除了AS25-S-寡核苷酸和AS1249-S-寡核苷酸外，还证明了AS-S-寡核苷酸对IL-10产生的抑制作用与不仅通过最近邻法计算的Tm值而且与通过吸光度对温度曲线确定的Tm值之间存在强正相关。