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

项目总结 靶向失调的蛋白激酶的小分子的发展导致了 癌症的治疗。然而，选择性证明需要新的方法来抑制激酶。 挑战和抗药性的出现。这项提案探索了腺苷如何编辑RNA 作用于RNA(ADAR)酶的脱氨酶可用于调节癌症中的靶蛋白活性，通过 优化引导寡核苷酸，促进在特定序列背景下的编辑。阿达尔家族 的酶将腺苷转化为核糖核酸中的肌苷，并可通过一种 互补引导寡核苷酸。到目前为止，ADAR酶的RNA编辑领域一直专注于 由于ADAR对编辑腺苷的偏好，纠正致病的提前终止密码子 在终止密码子中。然而，已知赖氨酸密码子会被ADAR去氨基，并且具有特殊的 感兴趣的原因是蛋白激酶中存在保守的催化赖氨酸残基。赖氨酸的反应 带有ADAR2的密码子将产生精氨酸密码子：一种已知的蛋白激酶失活突变。开车 在激酶mRNA中编辑催化赖氨酸，一种既定的合理设计方法来指导 将使用寡核苷酸化学修饰。我们的站点定向RNA编辑策略很高 治疗潜力，因为它利用内源性ADAR酶，因此只需要传递 一种指导性寡核苷酸。虽然酶的传递在效率和免疫刺激方面面临障碍， 寡核苷酸疗法最近在靶向递送方面取得了成功。这个项目的中心假设是 引导寡核苷酸对蛋白激酶mRNA进行编辑将导致下游细胞减少 癌症的信号。目标1将定义指南RNA修饰如何影响赖氨酸中目标腺苷的编辑 通过寡核苷酸化学合成和体外活性测定。目标2将评估蛋白质的效果 蛋白激酶信使核糖核酸编辑对胶质母细胞瘤细胞蛋白磷酸化和细胞凋亡的影响。细胞培养方面的培训， Western blotting和荧光显微镜将使细胞分析蛋白激酶mRNA编辑成为可能。 这项原则性实验的证明将通过确定疗效而在癌症治疗中产生广泛的影响。 RNA编辑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 ADAR编辑的应用，通过定义如何将引导RNA设计为以非首选顺序起作用 上下文。

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.