Arzumanov Andrey, Stetsenko Dmitry A, Malakhov Andrey D, Reichelt Stefanie, Sørensen Mads D, Babu B Ravindra, Wengel Jesper, Gait Michael J
Medical Research Council, Laboratory of Molecular Biology, Cambridge CB2 2QH, UK.
Oligonucleotides. 2003;13(6):435-53. doi: 10.1089/154545703322860762.
The HIV-1 trans-activation responsive element (TAR) RNA stem-loop interacts with the HIV trans-activator protein Tat and other cellular factors to stimulate transcriptional elongation from the viral long terminal repeat (LTR). Inhibitors of these interactions block full-length transcription and, hence, would potentially inhibit HIV replication. We have studied structure-activity relationships in inhibition of trans-activation by steric block 2'-O-methyl (OMe) oligonucleotides chimeras (mixmers) containing locked nucleic acid (LNA) units. Inhibition was measured both in Tat-dependent in vitro transcription from an HIV-1 DNA template directed by HeLa cell nuclear extract and in a robust HeLa cell reporter assay that involves use of stably integrated plasmids to express firefly luciferase Tat dependently and Renilla luciferase Tat-independently. OMe oligonucleotides with optimally 40%-50% LNA units and a minimum of 12 residues in length were active in the cellular assay when delivered with cationic gemini surfactant GS11 at 50% inhibitory concentrations of 230 +/- 40 nM, whereas activity in the in vitro transcription assay was observed down to 9 residues. No cellular activity was observed for OMe oligonucleotides of 12 or 16 residues, which was shown to be due to poor cellular uptake. Both 12-mer mixmers containing alpha -L-LNA or 2'-thio-LNA (S-LNA) were also active in in vitro transcription and the former in cellular reporter inhibition assays, demonstrating that the property of promotion of cellular uptake by LNA is not due to specific sugar conformational effects. Covalent conjugates of OMe/LNA chimeras with Kaposi-fibroblast growth factor (K-FGF) or Transportan peptides failed to enter HeLa cells without a delivery agent but were fully active when delivered by cationic gemini surfactant, showing that in principle, peptide conjugation does not interfere with cellular activity. Thus, OMe/LNA mixmers are powerful reagents for use as steric block inhibitors of gene expression regulated by protein-RNA interactions within HeLa cell nuclei.
HIV-1反式激活应答元件（TAR）RNA茎环与HIV反式激活蛋白Tat及其他细胞因子相互作用，以刺激病毒长末端重复序列（LTR）的转录延伸。这些相互作用的抑制剂会阻断全长转录，因此可能抑制HIV复制。我们研究了含有锁核酸（LNA）单元的空间位阻2'-O-甲基（OMe）寡核苷酸嵌合体（混合体）抑制反式激活的构效关系。在由HeLa细胞核提取物指导的HIV-1 DNA模板的Tat依赖性体外转录中以及在一个强大的HeLa细胞报告基因检测中测量抑制作用，该检测涉及使用稳定整合的质粒以Tat依赖性方式表达萤火虫荧光素酶和以Tat非依赖性方式表达海肾荧光素酶。当与阳离子双子表面活性剂GS11一起以230±40 nM的50%抑制浓度递送时，具有最佳40%-50% LNA单元且长度至少为12个残基的OMe寡核苷酸在细胞检测中具有活性，而在体外转录检测中观察到活性低至9个残基。对于12或16个残基的OMe寡核苷酸未观察到细胞活性，这表明这是由于细胞摄取不良所致。含有α-L-LNA或2'-硫代-LNA（S-LNA）的12聚体混合体在体外转录中也具有活性，前者在细胞报告基因抑制检测中具有活性，表明LNA促进细胞摄取的特性并非由于特定的糖构象效应。OMe/LNA嵌合体与卡波西成纤维细胞生长因子（K-FGF）或转运蛋白肽的共价缀合物在没有递送剂的情况下无法进入HeLa细胞，但在由阳离子双子表面活性剂递送时具有完全活性，表明原则上，肽缀合不会干扰细胞活性。因此，OMe/LNA混合体是用于作为HeLa细胞核内蛋白质-RNA相互作用调节的基因表达的空间位阻抑制剂的强大试剂。
