Identifying and Studying RNA Loop-Small Molecule Interactions

描述（由申请人提供）：RNA是设计小分子治疗药物和功能探针的重要靶标。然而，由于关于RNA基序如何与小分子相互作用的信息有限，它是一个未充分利用的靶标。本提案描述了识别RNA基序-小分子伙伴的研究，并研究了RNA和小分子中控制分子识别的重要特征。所使用的RNA基序是小的内部和发夹环，通常存在于生物学上重要的RNA中，如pre- microrna，未开发的和重要的RNA药物靶标，具有与我们的RNA基序相似的二级结构，没有已知的三级结构。两种协同方法被用来使平行探测RNA和小分子的特征。第一种方法是2D组合筛选（2DCS），同时筛选两个文库（一个阵列固定的小分子文库和一个RNA文库）来鉴定RNA基序小分子伴侣。从配体功能化的位置直接从阵列上获取rna，进行克隆和测序。选定的RNA和RNA基序-小分子复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2DCS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克隆，每个测序反应产生更多的序列数据。如果关于RNA-小分子相互作用的信息是可用的，一个RNA靶标的二级结构可以搜索一个或几个基序，其中一个小分子伴侣被识别，并且小分子“模块”定制链接以容纳目标RNA中的每个位点。这可能消除了对每个新RNA靶点进行高通量筛选分析的需要。本研究的具体目的是：1.)表征6'- n -5-己糖酸卡那霉素A的分子识别的内部环与AC对，共识环确定从先前的完全选择。交流电环以trna突变的形式存在，导致疾病。2)。鉴定和研究选择结合氨基糖苷库成员的RNA内环。3)。使用StARTS和2DCS简化鉴定和统计分析结合6'- n -5-己酸neamine的内部环。4)。扩展目标2和3中的结果以鉴定RNA发夹环-氨基糖苷相互作用。5)。通过start和2DCS鉴定肽库与RNA内环和发夹环之间的相互作用。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相互作用来发挥其作用。拟议研究的目标是了解药物样分子如何与RNA相互作用，以设计治疗药物或RNA功能探针。

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.