Identifying and Studying RNA Loop-Small Molecule Interactions

描述(申请人提供)：RNA是设计小分子疗法和功能探针的重要靶点。然而，它是一个没有得到充分利用的目标，因为关于RNA基序如何与小分子相互作用的信息有限。这项建议描述了识别RNA基序-小分子伙伴的研究，并调查管理分子识别的RNA和小分子中的重要特征。所使用的RNA基序是小的内部和发夹环，通常存在于生物重要的RNA中，如前microRNAs、未被充分利用的和重要的RNA药物靶标，其二级结构类似于我们的RNA基序，但没有已知的三级结构。使用两种协同方法来实现对RNA和小分子中的特征的并行探测。第一种方法是2D组合筛选(2DCS)，它同时筛选两个文库(一个是阵列固定化的小分子文库，另一个是RNA文库)，以确定RNA基序-小分子伙伴。从配基功能化的位置直接从阵列上获取RNA，克隆并测序。选定的RNA和RNA基序-小分子络合物可以通过核磁共振和光学熔融实验来研究它们的结构、柔韧性和热力学稳定性。第二种方法，通过测序的结构活性关系(STARTS)，通过测序数据中RNA环的出现，从2DC的输出中分配相对结合亲和力。多个选定的序列连接在一起并被克隆，每一次测序反应产生更多的序列数据。如果有关于RNA-小分子相互作用的信息，就可以在RNA目标的二级结构中搜索一个或几个小分子伙伴被识别的基序，并定制小分子“模块”以适应目标RNA中的每个位置。这可能会消除对每个新的RNA靶标进行高通量筛选试验的需要。这项研究的具体目的是：1)用AC对的内部环表征6‘-N-5-己糖酸卡那霉素A的分子识别，该共识环由先前的完全选择确定。AC环是以tRNA突变的形式出现的，会导致疾病。2.)鉴定和研究选择用来结合氨基糖苷类文库成员的RNA内环。3.)使用START和2DC简化识别和统计分析结合6‘-N-5-己烯酸新胺的内环。4.)扩展AIMS 2和AIMS 3中的结果以确定RNA发夹环-氨基糖苷类相互作用。5.)通过STARTS和2DCs确定类肽文库与RNA内部环和发夹环之间的相互作用。与公共健康相关的RNA是一种与疾病状态相关的重要生物分子，然而，很少有药物通过与RNA相互作用来产生作用。这项拟议的研究的目标是了解类药物分子如何与RNA相互作用，以设计治疗方法或RNA功能的探针。

DESCRIPTION (provided by applicant): RNA is an important target for the design of small molecule therapeutics and probes of function. It is an underutilized target, however, because of the limited information available about how RNA motifs interact with small molecules. This proposal describes studies to identify RNA motif-small molecule partners and investigate important features in both the RNA and small molecule that govern molecular recognition. The RNA motifs used are small internal and hairpin loops that are commonly found in biologically important RNAs such as pre- microRNAs, underexploited and important RNA drug targets with secondary structures similar to our RNA motifs and no known tertiary structure. Two synergistic methods are used to enable parallel probing of features in the RNA and the small molecule. The first method, 2D Combinatorial Screening (2DCS), screens two libraries simultaneously (an array-immobilized small molecule library and an RNA library) to identify RNA motif-small molecule partners. RNAs are harvested directly off the array from ligand-functionalized positions, cloned, and sequenced. Selected RNAs and RNA motif-small molecule complexes can be studied by NMR and optical melting experiments to interrogate their structures, flexibilities, and thermodynamic stabilities. The second method, Structure Activity Relationships Through Sequencing (StARTS), assigns relative binding affinities from the output of 2DCS via the occurrence of RNA loops in sequencing data. Multiple selected sequences are ligated together and cloned, yielding more sequence data per single sequencing reaction. If information about RNA-small molecule interactions were available, an RNA target's secondary structure could be searched for one or several motifs to which a small molecule partner was identified, and the small molecule "modules" custom-linked to accommodate each site in the target RNA. This may eliminate the need to subject each new RNA target to a high throughput screening assay. The specific aims for this study are: 1.) Characterize the molecular recognition of 6'-N-5-hexynoate kanamycin A by internal loops with AC pairs, the consensus loop determined from a previously completely selection. AC loops are present as mutations in tRNAs that cause disease. 2.) Identify and study the RNA internal loops selected to bind members of an aminoglycoside library. 3.) Use StARTS and 2DCS to streamline identification and statistical analysis of the internal loops that bind 6'-N-5-hexynoate neamine. 4.) Expand the results in Aims 2 and 3 to identify RNA hairpin loop-aminoglycoside interactions. 5.) Identify interactions between a peptoid library and RNA internal loops and hairpin loops via StARTS and 2DCS. PUBLIC HEALTH RELEVANCE RNA is an important biomolecule that is associated with diseased states, however, very few drugs elicit their effects by interacting with RNA. The goal of the proposed research is to understand how drug-like molecules interact with RNA to design therapeutics or probes of RNA function.