Developing Reliable In Silico Methods to Design Small Molecules Targeting RNA

描述（由申请人提供）：RNA具有重要而多样的生物学功能，包括催化、基因调控和细胞定位。功能异常会导致癌症、囊性纤维化和肌肉萎缩症等疾病。综上所述，RNA是小分子干预的重要治疗靶点。由于基因组测序的努力，新的RNA靶点被发现的速度正在增加。相比之下，设计或筛选结合和调节RNA功能的配体的方法远远落后。鉴定结合RNA的小分子最常用的方法是高通量筛选。不幸的是，RNA靶点的命中率远低于蛋白质。这在一定程度上是由于缺乏对小分子的特征的了解，这些特征预先决定了它们与RNA的结合，缺乏对RNA可靶向空间的了解，这极大地阻碍了RNA治疗学和RNA化学生物学领域的发展。提出的工作目标是开发计算方法来识别基因组序列和二级结构中的可药物RNA靶点，并合理设计小分子配体来靶向它们。该提案的具体目标是：(1)建立一种计算方法来识别可以用小分子靶向的rna。一个)。构建一个可搜索数据库的RNA基序-配体相互作用，已经确定。该数据库将使用开源的MySQL关系引擎构建，并可通过命令行界面和web访问；b。)开发一个搜索RNA二级结构的计算机程序，以识别数据库中具有两个或多个结合配体的基序的RNA结构域；c。)更新可搜索的数据库，因为新的信息小分子靶向RNA在文献中变得可用。通过网络上的服务器提供对软件的开放访问。(2)使用这些计算工具搜索所有人类pri- and pre- microrna的二级结构是否与Aim 1中描述的数据库重叠。基于这种重叠，配体将被设计成结合感兴趣的microRNA中存在的两个或多个基序，并将使用模块化组装方法合成，以提高靶标亲和力和特异性。(3)使用生化技术测试单体和模块化组装的配体靶向它们设计结合的microRNA。使用双荧光素酶报告系统确定在总细胞RNA存在和不存在的情况下配体结合位点和配体效力。项目描述第6页

DESCRIPTION (provided by applicant): RNA has essential and diverse biological functions including catalysis, gene regulation, and cellular localization. Aberrant function can lead to diseases such as cancer, cystic fibrosis, and muscular dystrophy. Taken together, RNA is an important therapeutic target for small molecule intervention. The rate at which new RNA targets are being discovered is increasing due to genomic sequencing efforts. In contrast, methods to design or screen ligands for binding to and modulating RNA function are lagging far behind. The most common method employed to identify small molecules that bind RNA is high throughput screening. Unfortunately, the hit rates for RNA targets are much lower than proteins. This is in part due to the lack of understanding about the features in small molecules that pre-dispose them for binding RNA and a lack of understanding about the RNA space that is targetable, significantly hampering developments in the fields of RNA therapeutics and RNA chemical biology. The goal of the proposed work is to develop computational methods to identify druggable RNA targets in genomic sequences and secondary structures and to rationally design small molecule ligands to target them. The specific aims of the proposal are: (1) Establish a computational approach to identify RNAs that can be targeted with a small molecule. a.) Construct a searchable database of RNA motif-ligand interactions that have been previously identified. The database will be constructed using the open-source MySQL relational engine and will be accessible via a command-line interface and the web; b.) Develop a computer program that searches RNA secondary structures to identify RNA domains that have two or more motifs that bind a ligand in the database; c.) Update the searchable database as new information on small molecules targeting RNA becomes available in the literature. Provide open access to the software via a server on the web. (2) Use these computational tools to search the secondary structures of all human pri- and pre- microRNAs for overlap with the database described in Aim 1. Based on this overlap, ligands will be designed that bind two or more motifs present in a microRNA of interest and will be synthesized using a modular assembly approach in order to enhance target affinity and specificity. (3) Test monomeric and modularly assembled ligands for targeting the microRNA to which they were designed to bind using biochemical techniques. Determine the ligand binding site in the presence and absence of total cellular RNA and ligand potency in vivo using a dual luciferase reporter system. Project Description Page 6