Development of method for analysis of long non-coding RNA by combination of DNAzyme, solid-phase DNA probe and mass spectrometry

2019 Fiscal Year Final Research Report

• Project/Area Number: 18K19302
• Research Category: Grant-in-Aid for Challenging Research (Exploratory)
• Allocation Type: Multi-year Fund
• Review Section: Medium-sized Section 43:Biology at molecular to cellular levels, and related fields
• Research Institution: Ehime University
• Principal Investigator: HORI Hiroyuki 愛媛大学, 理工学研究科(工学系), 教授 (20256960)
• Co-Investigator(Kenkyū-buntansha): 横川 隆志 岐阜大学, 工学部, 教授 (90242304)
• Project Period (FY): 2018-06-29 – 2020-03-31
• Keywords: 核酸 / 酵素 / RNA

Outline of Final Research Achievements

In RNA sequencing with modified nucleosides, the most reliable method is mass spectrometry. In general, the target RNA is digested with ribonuclease (RNase) and then the fragments are analyzed by mass spectrometry. In the case of long non-coding RNAs, however, cleavage by ribonucleases often generates duplicated RNA fragments, which possess the same sequence. Therefore, the difficulty of analysis of long non-coding RNAs by MS spectrometry rises with increasing length of RNA. To overcome this problem, we developed a method for analysis of long non-coding RNA. In the method, long non-coding RNA is site-specifically cleaved by deoxyribozyme (DNAzyme) and then its fragments are purified by the solid-phase DNA probe method. The sequence of RNA fragment is determined by mass spectrometry.

Free Research Field

機能生物化学

Academic Significance and Societal Importance of the Research Achievements

RNA中には、メチル化されたものやアセチル化されたものなど、様々な修飾ヌクレオシドが含まれます。これら修飾ヌクレオシドは、タンパク質合成系を制御し、発生・分化、発がん、感染、免疫など高次生命現象ともリンクします。従って、どのようなRNAの、どこに、どんな修飾ヌクレオシドが、どのくらいの頻度で存在するのかを調べるのは、現代の生命科学で重要な課題となっています。しかしながら、既存の手法のみで、長鎖RNAの修飾を調べるのは容易ではありません。本研究では、新たな手法を開発しました。この研究成果は、単に分子生物学・生化学分野のみにとどまらず、医学、薬学、農学、理学、工学等、多方面に波及するものです。