A high-throughput kit for detection of RNA modifications

描述(申请人提供)：该项目的总体目标是开发一种集成的湿/干实验室试剂盒，用于高通量检测RNA修饰。最终产品将提供所有必需的分子生物学试剂，一套简单而强大的实验操作，用于轻松分析修饰，以及研究人员友好的基于云的分析软件，不需要特殊的生物信息学技能或计算硬件。新出现的数据表明，许多类型的细胞RNA的转录后修饰在生物学以及神经系统的发育和维持中发挥着重要的、在很大程度上尚未被探索的作用。特别值得注意的是，许多神经疾病是由于RNA修饰的特定缺陷引起的，包括常染色体隐性智力残疾(ARID)、非综合征性X连锁智力低下、肌阵挛癫痫粗糙红纤维(MERRF)、线粒体脑病、伴有卒中样发作的乳酸酸中毒(MELAS)和家族性自主神经功能障碍。初步证据表明，RNA修饰调控与基因表达、mRNA剪接、核输出和蛋白质翻译之间的联系表明，由于大多数细胞类型中大多数RNA的修饰状态还没有很好地定义，因此可能会产生更广泛的未知影响。高通量RNA测序使基因表达研究发生了革命性的变化，但由于缺乏检测适合商业使用的修饰的方法，以及缺乏复杂的测序后分析方法，高通量RNA测序尚未广泛应用于鉴定大多数类型的RNA修饰。这一阶段1的建议集中于为三种类型的生物相关但相对研究较少的RNA修饰开发这样的方法学，具体目标如下：1.开发识别具有M1A、M3C或2‘-O-甲基化的RNA位点的方法。开发一条分析管道，以确定带有2‘-O-甲基化、M1a或M3c的位点。为了实现这些目标，将使用一种新的生化方法来检测和定位2‘-O-甲基化，利用已知的化学反应和连接方法来丰富这些位点。将采用一种独特的、新颖的方法 通过对酶处理的样品进行逆转录差异分析，检测M1a和M3c的修饰。将开发一个集成的、基于云的平台，带有定制的分析管道，用于处理和解释RNA测序数据，以简化准确识别修改所需的复杂分析。总而言之，这些包罗万象的试剂盒将形成用于大规模RNA修饰检测的测序前和测序后手段，并且易于使用。

DESCRIPTION (provided by applicant): The broad objective of this project is to develop an integrated wet-lab / dry-lab kit for high-throughput detection of RNA modifications. The final product will provide all required molecular biology reagents, a set of simple and robust experimental manipulations for facile analysis of modifications, and researcher-friendly analytic cloud-based software, requiring no special bioinformatics skills or computing hardware. Emerging data suggests that post-transcriptional modifications of many types of cellular RNA play an important and largely unexplored role in biology and in development and maintenance of the nervous system. In particular, a notable variety of neural diseases are due to specific defects in RNA modification, including autosomal recessive intellectual disability (ARID), non-syndromic X-linked mental retardation, myoclonus epilepsy ragged-red fibers (MERRF), mitochondrial encephalopathy lactic acidosis with stroke-like episodes (MELAS), and familial dysautonomia. Initial evidence for the links between RNA modification regulation and gene expression, mRNA splicing, nuclear export, and protein translation suggest much broader unknown effects given that modification states for most RNAs in most cell types are not well-defined. High-throughput RNA sequencing has revolutionized the study of gene expression, but it has not been widely applied to identifying most types of RNA modifications due to lack of methods for detection of modifications that are suitable for commercial use, and lack of methods for complex post-sequencing analytics. This Phase 1 proposal focuses on developing such methodology for three types of biologically relevant but relatively understudied RNA modifications, with the following Specific Aims: I. Develop methods for identifying RNA sites with m1A, m3C, or 2'-O-methylation. II. Develop an analysis pipeline to identify sites with 2'-O-methylation, m1A, or m3C. To achieve these aims, a novel biochemical approach will be employed for detecting and mapping 2'-O- methylation leveraging a known chemical reaction coupled with ligation methods to enrich for these sites. A distinct, novel approach will be applied to detect m1A, and m3C modifications, by differential analysis of reverse transcription between enzymatically treated samples. An integrated, cloud-based platform with custom analytic pipelines will be developed for processing and interpreting the RNA sequencing data to simplify complex analyses required to identify modifications with precision. Together, these all-inclusive kits will form both pre- and post-sequencing means for large-scale RNA modification detection with turn-key ease of use.