RNA Decoys for DNA Binding Proteins

越来越多的人认识到小的非编码RNA可以参与基因调控。小RNA能抑制DNA结合蛋白吗？我们已经开发了一个人工的例子，通过进行体外遗传选择实验，确定了一个小的RNA适体，竞争性地抑制人转录因子NF-κ B结合到DNA在体外。通过酵母体内遗传选择的优化导致在活酵母细胞中抑制NF-κ B的RNA。我们已经解决了这种不寻常的RNA/NF-Ⅰ <B复合物的X射线共晶结构。这种结构证明了RNA结构的可塑性如何允许DNA模仿，提出了一种新的策略，用于工程化RNA特异性针对不同的NF-κ B二聚体，并确定了一种新的方法。 新的“开放”NF-Ⅰ B构象与DNA结合不相容。NF-κ B在调节炎症反应相关基因中起重要作用，并保护肿瘤细胞免于凋亡，从而限制化疗和放疗的有效性。降低NF-κ B活性的药物倾向于促进细胞凋亡，并显示出有希望的抗癌活性。使用NF-κ B作为治疗感兴趣的模型，我们将在细菌，酵母和人类细胞培养实验中优化和测试抗NF-κ B RNA适体。我们还将确定游离RNA适体的结构，以了解蛋白质结合后发生的构象变化。我们前所未有的RNA/NF-κ B晶体结构现在允许虚拟药物 筛选和体外和体内验证测定新的NF-κ B抑制剂。这些药物将通过稳定我们新发现的与DNA结合不相容的“开放”NF-κ B构象来发挥作用。 提出了四个具体目标： 目标1.通过在细菌和酵母中的遗传选择优化和扩展抗NF-κ B RNA适体功能。 目标二。分析抗NF-κ B RNA适体结合NF-κ B的结构基础。 目标3：在培养的哺乳动物细胞中分析抗NF-κ B RNA适体对NF-κ B的抑制。 目标4。鉴定抑制NF-κ B与DNA结合的小分子。

There is growing appreciation that small, non-coding RNAs can participate in gene regulation. Can small RNAs inhibit DNA-binding proteins? We have developed an artificial example by performing in vitro genetic selection experiments identifying a small RNA aptamer that competitively inhibits human transcription factor NF-kappaB binding to DNA in vitro. Optimization by yeast in vivo genetic selections resulted in an RNA that inhibits NF-kappaB in living yeast cells. We have solved the X-ray co-crystal structure of this unusual RNA/NF-I<B complex. This structure demonstrates how RNA structural plasticity allows DNA mimicry, suggests a new strategy for engineering RNAs specific for different NF-kappaB dimers, and identifies a novel "open" NF-I<B conformation incompatible with DNA binding. NF-kappaB plays an essential role in regulating genes involved in inflammatory responses and protects tumor cells from apoptosis, thus limiting the effectiveness of chemotherapy and radiotherapy. Agents that reduce NF-kappaB activity tend to promote apoptosis and have shown promising anticancer activities. Using NF-kappaB as a model of therapeutic interest, we will optimize and test anti-NF-kappaB RNA aptamers in bacterial, yeast, and human cell culture experiments. We will also determine the structure of the free RNA aptamer to understand conformational changes that occur upon protein binding. Our unprecedented RNA/NF-kappaB crystal structure now allows virtual drug screens and in vitro and in vivo validation assays for novel NF-KappaB inhibitors. Such drugs would act by stabilizing our newly-discovered "open" NF-kappaB conformation that is incompatible with DNA binding. Four specific aims are proposed: Aim 1. Optimize and extend anti-NF-kappaB RNA aptamer function by genetic selections in bacteria and yeast. Aim 2. Analyze the structural basis for NF-kappaB binding by anti-NF-kappaB RNA aptamers. Aim 3. Analyze NF-kappaB inhibition by anti-NF-kappaB RNA aptamers in cultured mammalian cells. Aim 4. Identify small molecules that inhibit DNA binding by NF-kappaB.