Site-directed RNA Editing to Modulate Kinase Activity as a Chemotherapeutic

项目摘要 靶向失调蛋白激酶的小分子的发展导致了蛋白激酶的范式转变。 癌症的治疗然而，需要新的激酶抑制方法是由选择性证明的 挑战和耐药性的出现。该提案探讨了腺苷如何编辑RNA 作用于RNA的脱氨酶（阿达尔）酶可用于调节癌症中的靶蛋白活性， 优化在特定序列环境中促进编辑的指导寡核苷酸。阿达尔家族 的酶将腺苷转化为RNA中的肌苷，并且可以通过靶向腺苷， 互补引导寡核苷酸。到目前为止，通过阿达尔酶进行RNA编辑的领域集中在 由于阿达尔对编辑腺苷的偏好，校正致病性提前终止密码子 在终止密码子内。然而，已知赖氨酸密码子被ADAR脱氨基，并且特别是在氨基酸序列中。 由于蛋白激酶中存在保守的催化赖氨酸残基，赖氨酸的反应 密码子与ADAR 2的结合会产生精氨酸密码子：一种已知的蛋白激酶失活突变。开车 在激酶mRNA内的催化赖氨酸处进行编辑，这是一种已建立的合理设计方法， 将使用寡核苷酸化学修饰。我们的定点RNA编辑策略具有高 治疗潜力，因为它利用内源性阿达尔酶，因此仅需要递送 指导寡核苷酸。虽然酶递送面临效率和免疫刺激方面的障碍， 寡核苷酸疗法最近在靶向递送方面取得了成功。这个项目的核心假设是 蛋白激酶mRNA指导性核苷酸定向编辑将导致下游细胞的减少 癌症中的信号。目的1将定义指导RNA修饰如何影响赖氨酸中靶腺苷的编辑 密码子通过寡核苷酸化学合成和体外活性测定。目的2将评估蛋白质的效果 激酶mRNA编辑对胶质母细胞瘤细胞中蛋白磷酸化和凋亡的影响。细胞培养培训， Western印迹和荧光显微镜将使蛋白激酶mRNA编辑的细胞分析成为可能。 这一原理性实验的证明将通过建立有效性而在癌症治疗中具有广泛的意义。 来调节无序的蛋白质活性。更广泛地说，这将扩大潜在的治疗 通过定义引导RNA如何被工程化以在非优选序列中起作用来应用阿达尔编辑 contexts.

PROJECT SUMMARY The development of small molecules that target dysregulated protein kinases led to a paradigm shift in the treatment of cancer. However, the need for novel methods of kinase inhibition is demonstrated by selectivity challenges and the emergence of drug resistance. This proposal explores how RNA editing by Adenosine Deaminase Acting on RNA (ADAR) enzymes can be used to modulate target protein activity in cancers, through the optimization of guide oligonucleotides that promote editing in specific sequence contexts. The ADAR family of enzymes converts adenosine to inosine in RNA, and can be directed to a target adenosine by a complementary guide oligonucleotide. Thus far, the field of RNA editing by ADAR enzymes has focused on correcting disease-causative premature termination codons, due to ADAR’s preference for editing adenosines within a termination codon. However, lysine codons are known to be deaminated by ADARs, and are of particular interest due to the presence of a conserved catalytic lysine residue in protein kinases. Reaction of the lysine codon with ADAR2 would produce an arginine codon: a known inactivating mutation in protein kinases. To drive editing at the catalytic lysine within kinase mRNA, an established rational design approach to guide oligonucleotide chemical modifications will be used. Our strategy for site-directed RNA editing has high therapeutic potential because it utilizes endogenous ADAR enzymes, and therefore only requires the delivery of a guide oligonucleotide. While enzyme delivery faces barriers in efficiency and immune stimulation, oligonucleotide therapies have had recent successes in targeted delivery. The central hypothesis of this project is that guide oligonucleotide-directed editing of protein kinase mRNA will lead to a reduction in downstream cell signaling in cancers. Aim 1 will define how guide RNA modifications affect editing of target adenosines in lysine codons via oligonucleotide chemical synthesis, and in vitro activity assays. Aim 2 will evaluate the effect of protein kinase mRNA editing on protein phosphorylation and apoptosis in glioblastoma cells. Training in cell culture, Western blotting, and fluorescence microscopy will enable the cellular analysis of protein kinase mRNA editing. This proof of principle experiment will have broad implications in cancer therapeutics by establishing the efficacy of RNA editing to modulate disordered protein activity. More broadly this will expand the potential therapeutic applications of ADAR editing by defining how guide RNAs can be engineered to act in non-preferred sequence contexts.