Identifying and Studying RNA Loop-Small Molecule Interactions

识别和研究 RNA 环-小分子相互作用

• 批准号: 7533874
• 负责人: Matthew D Disney
• 金额: $ 29.81万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7533874
• 关键词: 2-Aminopurine; Affect; Affinity; Aminoglycosides; Base Pairing; Binding; Binding Sites; Biological; Biological Assay; Biological Models; Charge; Chemicals; Classification; Complex; Consensus; Custom; Data; Defect; Disease; Drug Delivery Systems; Fluorescence; Foundations; Genomics; Goals; Harvest; Investigation; Kanamycin A; Label; Lead; Libraries; Ligand Binding; Ligands; Link; Methods; MicroRNAs; Mitochondria; Mutation; NMR Spectroscopy; Neomycin; Nucleotides; Oligonucleotides; Optics; Output; Pattern; Peptoids; Pharmaceutical Preparations; Pliability; Positioning Attribute; Public Health; RNA; RNA Binding; RNA Probes; RNA Sequences; RNA library; Rate; Reaction; Relative (related person); Research; Sampling; Screening procedure; Shapes; Site; Structure; Structure-Activity Relationship; Surface; Tail; Therapeutic; Thermodynamics; Tobramycin; aptamer; base; combinatorial; design; functional group; high throughput screening; improved; melting; member; molecular recognition; preference; research study; size; small molecule; small molecule libraries; stem; two-dimensional

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.

描述（由申请人提供）：RNA是设计小分子疗法和功能探针的重要目标。但是，由于可用的有关RNA图案如何与小分子相互作用的信息有限，因此它是一个未充分利用的靶标。该建议描述了鉴定RNA基序的分子伙伴的研究，并研究控制分子识别的RNA和小分子中的重要特征。所使用的RNA基序是在生物学上重要的RNA中常见的小内部和发夹环，例如前microRNA，含量不足和重要的RNA药物靶标的二级结构类似于我们的RNA基序，没有已知的第三纪结构。两种协同方法用于实现RNA和小分子中特征的平行探测。第一种方法是2D组合筛选（2DC），同时筛选了两个库（一个阵列iMmobimized的小分子库和一个RNA库），以识别RNA图案小分子分子伙伴。将RNA直接从阵列中从配体功能化位置上收获，克隆和测序。可以通过NMR和光学融化实验研究选定的RNA和RNA基序分子复合物，以询问其结构，灵活性和热力学稳定性。第二种方法是通过测序（开始）通过测序的结构活动关系，通过在测序数据中出现RNA循环从2DC的输出中分配相对的结合亲和力。将多个选定的序列连接在一起并克隆在一起，每单个测序反应产生更多的序列数据。如果提供了有关RNA-MALL分子相互作用的信息，则可以搜索RNA靶标的二级结构，以找到一个或几个基序，并鉴定出一个小分子伴侣，并定制了小分子“模块”以容纳目标RNA中的每个位点。这可能消除了将每个新的RNA目标进行高吞吐量筛选测定法的需求。这项研究的具体目的是：1。）表征通过与交流对的内部环的分子识别6'-N-5-己酸卡纳米霉素A，这是根据先前完全选择确定的共识环。交流环是引起疾病的TRNA中的突变。 2.）识别和研究选择结合氨基糖苷库的成员的RNA内环。 3.）使用启动和2DC来简化结合6'-N-5-HEXYNOATE NEAMINE的内部环的识别和统计分析。 4.）将结果扩展到目标2和3中，以识别RNA发夹环 - 氨基糖苷相互作用。 5.）通过启动和2DC确定肽库和RNA内部环与发夹环之间的相互作用。公共卫生相关性RNA是与患病状态相关的重要生物分子，但是，很少有药物通过与RNA相互作用引起其作用。拟议的研究的目的是了解药物样分子如何与RNA相互作用以设计治疗剂或RNA功能探针。