用计算机软件辅助设计和实验分析筛选有效的反义核酸

目的用RNAstrueture软件设计和实验分析来筛选有效的反义核酸。方法选择Bel-2基因为靶基因,利用RNAstrueture软件,模拟Bcl-2mRNA的二级结构,根据自由能不稳定区域或者无折叠的区域,设计5条反义核酸序列,研究它们对白血病细胞的细胞生长作用,用免疫组织化学和流式细胞技术研究蛋白的表达,RT-PCR检测mRNA的含量,有细胞形态学方法、电泳和流式细胞技术检测细胞凋亡。结果5条中有两条反义核酸能显著抑制白血病细胞的生长,降低Bcl-2mRNA和蛋白水平,显著诱导细胞凋亡。结论计算机软件预测有效反义比其他方法快而有效,这种方法能有效加速研究和临床反义序列的设计。

Selection of Effective Bcl-2 Antisense Oligodeoxynucleotides with Computer Software and Experimental Assay

Objective: To explore and investigate the selection of effective antisense oligodeoxynuleotides with the help of computer and RNAstructure folding software. Methods: Bcl-2 gene was used as the target gene and five antisense oligodeoxynuleotides were designed to be bound to Bcl-2 mRNA optimal secondary structure regions that were predicted free from intramolecular fold or instability of free energy. The five antisense oligodeoxynucleotides were studied with experimental assay of leukemia cells, including cell grow assay with tropan blue exclusion, expression of Bcl-2 protein detected with immunochemistry and flowcytometry, Bcl-2 mRNA content detected with RT-PCR technique, as well as apoptosis observed and determined with morphonological method, electrophoresis and flowcytometry. Results: The results showed that two of the five antisense oligodeoxynucleotides were effective antisense oligodeoxynucleotides, which were able to inhibit cell growth in leukemia, to decrease the level of Bcl-2 mRNA and protein, to induce apoptosis of leukemia cells significantly. Conclusion: The computational prediction of antisense efficacy is faster than other methods and more efficient, which can potentially speed the development of sequences for both research and clinical applications.