amiRNA分子设计及其离体合成策略

详细介绍了基于WMD3 (Web MicroRNA Designer 3) 软件平台的amiRNA (artificial microRNA)分子自动设计方法及其离体合成策略。应用网络在线设计时，只需输入目的基因靶序列相关信息后便可获得候选amiRNA。根据选定的最佳amiRNA，可得到四条含有amiRNA以及载体中miRNA 两侧序列的寡聚核苷酸序列。重叠延伸PCR合成策略可以以这四条序列以及根据质粒模板设计的A、B两段序列作为引物，扩增出目标amiRNA。同样尿嘧啶切除的策略也可合成amiRNA，但这些引物序列需做适当调整变动。此外，本文还介绍了基于特异引物退火的amiRNA合成策略，该策略可保证amiRNA分子能够一步PCR合成，合成后的amiRNA表达盒可通过合适的限制性位点被克隆至目的载体中的相应位点。可以预见，这种amiRNA分子设计的科学性与合成策略的精确性将会使amiRNAi技术在生物基因功能分析中发挥更加重要的作用，对生命科学研究产生深远的影响。

Molecular Designing of amiRNA and its Synthesis Strategy in vitro

Detailedly described several reliable methods for constructing amiRNA , which was based on WMD3 (Web MicroRNA Designer 3), including molecular designing and flexible synthesis strategy in vitro. When designed online, four steps were needed sequentially: inputting target sequence(s), screening candidate amiRNAs, gaining four oligonucleotides carrying amiRNA-related sequence and other proper sequences that flanked nature miRNA. For overlapping PCR synthesis strategy, amiRNA can be amplified using these four oligonucleotides and two primers designed based on plasmid. Uracil-excision based strategy could also be employed to generate amiRNA, while primers need altering. Specific primer-annealing based method was equally represented here, which may guarantee the synthesis of amiRNA in one-step-PCR. With appropriate restriction sites, the synthesized amiRNA expression cassette can be cloned into corresponding sites of destination vector. It is foreseeable that such scientificness of molecular designing principle and the accuracy of synthesis strategy would aid greatly in gene functional analysis, promising a profound impact on life science.