Carrier-free cellular uptake and the gene-silencing activity of the lipophilic siRNAs is strongly affected by the length of the linker between siRNA and lipophilic group
作者:Natalya S. Petrova、Ivan V. Chernikov、Mariya I. Meschaninova、IIya S. Dovydenko、Aliya G. Venyaminova、Marina A. Zenkova、Valentin V. Vlassov、Elena L. Chernolovskaya
DOI:10.1093/nar/gkr1002
日期:2012.3
The conjugation of siRNA to molecules, which can be internalized into the cell via natural transport mechanisms, can result in the enhancement of siRNA cellular uptake. Herein, the carrier-free cellular uptake of nuclease-resistant anti-MDR1 siRNA equipped with lipophilic residues (cholesterol, lithocholic acid, oleyl alcohol and litocholic acid oleylamide) attached to the 5′-end of the sense strand via oligomethylene linker of various length was investigated. A convenient combination of H-phosphonate and phosphoramidite methods was developed for the synthesis of 5′-lipophilic conjugates of siRNAs. It was found that lipophilic siRNA are able to effectively penetrate into HEK293, HepG2 and KB-8-5 cancer cells when used in a micromolar concentration range. The efficiency of the uptake is dependent upon the type of lipophilic moiety, the length of the linker between the moiety and the siRNA and cell type. Among all the conjugates tested, the cholesterol-conjugated siRNAs with linkers containing from 6 to 10 carbon atoms demonstrate the optimal uptake and gene silencing properties: the shortening of the linker reduces the efficiency of the cellular uptake of siRNA conjugates, whereas the lengthening of the linker facilitates the uptake but retards the gene silencing effect and decreases the efficiency of the silencing.
通过与分子结合,这些分子可以通过自然运输机制进入细胞,从而增强siRNA的细胞摄取。在此,我们研究了通过不同长度的亚甲基连接子将疏水性残基(胆固醇、石胆酸、油醇和石胆酸油胺)连接到正义链的5′端,制备的耐核酸酶的抗MDR1 siRNA的无载体细胞摄取。我们开发了一种方便的H-磷酸盐和磷酰胺方法的组合,用于合成siRNA的5′-疏水性偶联物。发现当在微摩尔浓度范围内使用时,疏水性siRNA能够有效地渗透到HEK293、HepG2细胞和KB-8-5癌细胞中。摄取效率取决于疏水性部分类型、部分与siRNA之间的连接子长度以及细胞类型。在所有测试的偶联物中,含有6到10个碳原子的连接子连接的胆固醇化siRNA显示出最佳的摄取和基因沉默特性:连接子缩短会降低siRNA偶联物的细胞摄取效率,而连接子延长有助于摄取,但会延迟基因沉默效应并降低沉默效率。