Abstract：
Adenosine Deaminases Acting on RNA (ADARs) catalyze the deamination of adenosine to inosine in double-stranded RNA (dsRNA). ADARs\' ability to recognize and edit dsRNA is dependent on local sequence context surrounding the edited adenosine and the length of the duplex. A deeper understanding of how editing efficiency is affected by mismatches, loops, and bulges around the editing site would aid in the development of therapeutic gRNAs for ADAR-mediated site-directed RNA editing (SDRE). Here, a SELEX (systematic evolution of ligands by exponential enrichment) approach was employed to identify dsRNA substrates that bind to the deaminase domain of human ADAR2 (hADAR2d) with high affinity. A library of single-stranded RNAs was hybridized with a fixed-sequence target strand containing the nucleoside analog 8-azanebularine that mimics the adenosine deamination transition state. The presence of this nucleoside analog in the library biased the screen to identify hit sequences compatible with adenosine deamination at the site of 8-azanebularine modification. SELEX also identified non-duplex structural elements that supported editing at the target site while inhibiting editing at bystander sites.
摘要：
作用于RNA的腺苷脱氨酶(ADAR)催化双链RNA(dsRNA)中的腺苷脱氨基为肌苷。ADAR识别和编辑dsRNA的能力取决于编辑的腺苷周围的局部序列环境和双链体的长度。更深入地了解编辑效率如何受到不匹配的影响，循环,和围绕编辑位点的凸起将有助于ADAR介导的位点定向RNA编辑（SDRE）的治疗性gRNA的开发。这里,采用SELEX(通过指数富集的配体的系统进化)方法来鉴定以高亲和力与人ADAR2(hADAR2d)的脱氨酶结构域结合的dsRNA底物。将单链RNA文库与固定序列的靶链杂交，该靶链包含模拟腺苷脱氨过渡状态的核苷类似物8-氮杂bularine。该核苷类似物在文库中的存在使筛选偏向于鉴定与8-氮杂鸟嘌呤修饰位点处的腺苷脱氨基相容的命中序列。SELEX还鉴定了在目标位点支持编辑而在旁观者位点抑制编辑的非双链体结构元件。
