RNA Decoys for DNA Binding Proteins

人们越来越认识到，小的、非编码的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例子，通过进行体外基因选择实验，确定了一个小的RNA适配子，在体外竞争性地抑制人转录因子NF-kappaB与DNA的结合。酵母体内遗传选择的优化导致了抑制活的酵母细胞中的核因子-kappaB的RNA。我们已经解决了这种不同寻常的RNA/核因子-I&B络合物的X射线共晶结构。这种结构展示了RNA结构可塑性如何允许DNA模仿，提出了一种针对不同核因子-kappaB二聚体的工程RNA的新策略，并发现了一种 与DNA结合不相容的新的“开放”的核因子-I和B构象。核因子-kappaB在调节参与炎症反应的基因和保护肿瘤细胞免于凋亡方面发挥着重要作用，从而限制了化疗和放射治疗的效果。降低核因子-kappaB活性的药物往往会促进细胞凋亡，并显示出良好的抗癌活性。使用核因子-kappaB作为一个有治疗意义的模型，我们将在细菌、酵母和人类细胞培养实验中优化和测试抗核因子-kappaB RNA适体。我们还将确定游离RNA适配子的结构，以了解蛋白质结合时发生的构象变化。我们史无前例的RNA/核因子-kappaB晶体结构现在允许虚拟药物 新型核因子-kappaB抑制剂的筛选及体外和体内验证分析。这类药物将通过稳定我们新发现的与DNA结合不相容的“开放的”核因子-kappaB构象来发挥作用。 提出了四个具体目标： 目的1.通过在细菌和酵母中进行遗传选择，优化和扩展抗核因子-kappaB RNA适配子功能。 目的2.分析抗核因子-kappaB RNA适配子与核因子-kappaB结合的结构基础。 目的3.分析抗核因子-kappaB RNA适配子对培养的哺乳动物细胞中核因子-kappaB的抑制作用。 目的4.鉴定抑制核因子-kappaB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.