Biochemical analysis of the AGO-siRNA complex about DNA binding activity to induce the TGS (Transcriptional Gene Silencing).

我们的研究小组之前发表的数据表明，siRNA靶向未转录的HIV-1启动子区域的保守区域，单剂量的Prom a siRNA诱导了HIV复制的长期和深刻的抑制。通过几项试验，转录沉默（TGS）对HIV繁殖的抑制提供了明确的证据。越来越多的证据表明，siRNA结合蛋白Argonaute家族在siRNA诱导的TGS中起着关键作用。在TGS中，Ago1蛋白在沉默起始位点积累。为了探讨Ago1在siRNA诱导的HIV-1 TGS中的作用，我们建立了体外结合实验，检测Ago1 -siRNA复合物能否与靶DNA结合。该分析系统清楚地表明，这些复合物可以直接结合靶DNA序列。免疫染色法检测细胞感染HIV-1后，AGO1蛋白与siRNA共定位。我们还证明了这些TGS效应依赖于siRNA和靶序列之间的紧密匹配。

Our research group previously published data demonstrate that of siRNA targeting conserved regions of the untranscribed HIV-1 promoter region, a single dose of either Prom A siRNA induced prolonged and profound suppression of HIV re-production. Definitive evidence of transcriptional silencing (TGS) was provided to this repression of HIV reproduction by several assays. There is accumulating evidence the Argonaute family of siRNA binding proteins, play a pivotal role in siRNA induced TGS. In TGS Ago1 protein accumulates at the site of initiation of silencing. To explore the role of Ago1 in siRNA induced TGS of HIV-1, in vitro binding assay was established for detecting AGO1-siRNA complex can be bind to target DNA. This assay system clearly demonstrated that these complex canb ebind directly to taget DNA sequence. AGO1 protein was co-localized with siRNA when the cells infected HIV-1 by immune-staining assay. We also demonstrated that these TGS effects are dependent upon close matching between the siRNA and the target sequence.