IGF::OT::IGF Developing Novel Tools for Site-Specific Quantitative Analysis of RNA ModificationsPeriod Performance: September 19, 2017 through June 16, 2018

IGF::OT::IGF 开发用于 RNA 修饰位点特异性定量分析的新工具期间表现：2017 年 9 月 19 日至 2018 年 6 月 16 日

• 批准号: 9571681
• 负责人: Lian Tian
• 金额: $ 24.84万
• 依托单位: MEDIOMICS, LLC
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-19 至 2018-06-18
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9571681
• 关键词: Affinity; Antibodies; Antibody Affinity; Antigens; Biological; Biological Assay; Carrier Proteins; Chickens; Coupled; Detection; Development; Enzyme-Linked Immunosorbent Assay; Goals; IgY; Immunize; Keyhole Limpet Hemocyanin; Liquid Chromatography; Malignant Neoplasms; Mass Spectrum Analysis; Methods; Modification; Nucleosides; Performance; Phase; RNA; RNA analysis; Research; Research Personnel; Sampling; Schedule; Scientist; Series; Site; Site-Directed Mutagenesis; Specificity; Technology; Testing; Time; cost; cost effective; deep sequencing; improved; next generation sequencing; novel; polyclonal antibody; tool

Although recent developments in highly sensitive technologies have revolutionized research on RNA modifications, the available quantitative methods can be further improved. Current methods include liquid chromatography-tandem mass spectroscopy (LC-MS/MS) and next-generation sequencing coupled with modification-specific antibodies. The LC-MS/MS method is not accessible to many researcher scientists due to its high cost and specialization. Deep sequencing is only useful for research on specific RNA modifications if high quality antibodies are commercially available. We found there are at least a dozen RNA modifications that are not commercially available, but have been suggested to be related to cancer, suggesting a gap in the antibody market. Furthermore, though antibody pull-down followed by RT-qPCR can be used to evaluate the modification of a specific RNA, this method is not site-specific, and will not work well when there are multiple modification sites in one targeted RNA. There is a need for a simpler method to quantitatively analyze site-specific RNA modifications beyond the current options available. Here, we propose to 1) fill the gap in next-generation sequencing approach by developing high-affinity antibodies for cancer-related modifications with no commercially available antibodies, and 2) develop simpler alternative methods for the site-specific quantification of RNA modifications using Mediomics’ PINCER technology.

虽然最近高灵敏技术的发展使RNA修饰的研究发生了革命性的变化，但现有的定量方法还可以进一步改进。目前的方法包括液相色谱-串联质谱仪(LC-MS/MS)和结合修饰特异性抗体的下一代测序。由于LC-MS/MS方法的高成本和专属性，许多研究人员无法获得该方法。只有在商业上有高质量抗体的情况下，深度测序才对特定RNA修饰的研究有用。我们发现，至少有12种RNA修饰没有商业化，但被认为与癌症有关，这表明抗体市场存在缺口。此外，虽然抗体下拉和RT-qPCR可以用来评估特定RNA的修饰，但这种方法不是位点特异性的，当一个靶向RNA中有多个修饰位点时，这种方法不能很好地工作。除了目前可用的选择外，还需要一种更简单的方法来定量分析特定部位的RNA修饰。在这里，我们建议1)通过开发针对癌症相关修饰的高亲和力抗体来填补下一代测序方法的空白，而没有商业上可用的抗体，以及2)开发更简单的替代方法，使用Mediology的钳子技术来对RNA修饰进行位点特异性定量。