Shape RNA Structure Analysis for Drug Discovery and Translational Research

 描述(申请人提供)：大约70%的人类基因组被转录成RNA，在大多数这些RNA中，复杂的高阶结构是关键生物学过程的基础或根本影响。RNA结构是许多抗生素的靶标，在小分子药物开发中应进一步利用RNA结构。例如，像HIV和流感这样的RNA病毒基因组中的结构对病毒复制和致病至关重要。与人类疾病最密切相关的单核苷酸多态中，有一半发生在非编码区，可能与异常的RNA结构有关。许多基于核酸的疗法-包括临床上重要的反义和siRNA方法-只有在非常罕见、高亲和力和结构上可访问的部位成为靶点时才有效。Week实验室开发了一种名为SHAPE-MAP的化学探测策略，可以准确地报告生理相关环境中的RNA结构。这项技术使核苷酸分辨RNA结构分析信息能够通过大规模并行测序读出。尽管独立组织将SHAPE描述为RNA结构分析的“黄金标准”，但就其目前的形式而言，SHAPE-MAP代表了一种尖端但研究级的技术。Ribmetrix的长期愿景是使SHAPE-MAP成为一种在药物发现、转化研究和基础生物发现中应用的平台技术。在这项工作中，我们将进行概念验证研究，以解决非专家实验室采用SHAPE-MAP的障碍，目标有两个：(1)创建并通过实验验证诊断控制，以使非专家用户能够对原型SHAPE-MAP RNA结构分析管道进行有效的评估和故障排除；(2)检查和优化SHAPE试剂的配方，以实现一致的处理。数据显示，新手用户能够使用易于操作、寿命长的试剂实施和诊断RNA结构探测实验，与定量基准一致，这将证明进入第二阶段是合理的。在第二阶段，Ribometrix将自动化完全解释SHAPE-MAP实验所需的下游计算步骤，并将创建完整的工业化试剂平台，供制药和学术客户和合作者使用。用户友好的SHAPE技术平台的成功开发将使生物医学科学所有领域的研究人员能够进行强有力的定量实验，探索RNA在生物学中的基础作用，并促进基于RNA的和以RNA为导向的疗法的创建。

 DESCRIPTION (provided by applicant): Roughly 70% of the human genome is transcribed into RNA and, in most of these RNAs, complex higher-order structures underlie or fundamentally affect critical biological processes. RNA structures are the targets of many antibiotics, and RNA structure should be further exploited in small-molecule drug discovery efforts. For example, structures in the genomes of RNA viruses like HIV and influenza are essential for viral replication and pathogenesis. Half of the single nucleotide polymorphisms that are most strongly associated with human diseases occur in non-coding regions and are likely associated with abnormal RNA structures. Many nucleic acid-based therapeutics - including clinically important antisense and siRNA approaches - only work when exceedingly rare, high affinity, and structurally accessible sites are targeted. The Weeks laboratory has developed a chemical probing strategy, called SHAPE-MaP, that accurately reports on RNA structure in physiologically relevant contexts. This technology enables nucleotide-resolution RNA structure analysis information to be read out by massively parallel sequencing. Although independent groups describe SHAPE as the "gold standard" of RNA structure analysis, in its current form, SHAPE-MaP represents a cutting-edge but research-grade technology. The long-term vision of Ribometrix is to make SHAPE-MaP a platform technology with applications in drug discovery, translational research, and basic biological discovery. In this work, we will conduct proof-of-concept studies to solve impediments to adoption of SHAPE-MaP by non-expert laboratories via two aims: (1) to create and experimentally validate diagnostic controls to enable efficient evaluation and troubleshooting of a prototype SHAPE-MaP RNA structure analysis pipeline by non-expert users and (2) to examine and optimize formulation of SHAPE reagents for consistent handling. Progress to Phase 2 will be justified by data showing that novice users are able to implement and diagnose RNA structure probing experiments using easily handled, long-lifetime reagents, consistent with quantitative benchmarks. In Phase 2, Ribometrix will automate the downstream computational steps required to fully interpret SHAPE-MaP experiments and will create complete industrialized reagent platforms for use by pharmaceutical and academic customers and collaborators. Successful development of a user-friendly SHAPE technology platform will enable researchers in all areas of biomedical science to perform robust, quantitative experiments that explore the fundamental role of RNA in biology and that facilitate creation of RNA-based and RNA-directed therapeutics.