Development of aptamer recognizing native higher molecular structures of viroid – circular single-stranded RNA pathogen

Research Project

Project/Area Number	18K19196
Research Category	Grant-in-Aid for Challenging Research (Exploratory)
Allocation Type	Multi-year Fund
Review Section	Medium-sized Section 39:Agricultural and environmental biology and related fields
Research Institution	Hirosaki University
Principal Investigator	Sano Teruo 弘前大学, 農学生命科学部, 教授 (30142699)
Project Period (FY)	2018-06-29 – 2021-03-31
Project Status	Completed (Fiscal Year 2020)
Budget Amount *help	¥6,240,000 (Direct Cost: ¥4,800,000、Indirect Cost: ¥1,440,000) Fiscal Year 2020: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000) Fiscal Year 2019: ¥2,860,000 (Direct Cost: ¥2,200,000、Indirect Cost: ¥660,000) Fiscal Year 2018: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
Keywords	アプタマー / ウイロイド / 診断薬 / 防除薬 / SELEX / アンプリコン解析 / SELEX法
Outline of Final Research Achievements	Viroids are circular single-strand RNA pathogens of about 250-430 nucleotides. SELEX, systematic evolution of ligands by exponential enrichment, was carried out with the aim of selecting aptamers that can specifically bind to viroids. A variety of DNA sequences that bound to full-length potato spindle tuber viroid (PSTVd) RNA were enriched by 15 rounds of SELEX from a random pool of single-stranded 30-nucleotide DNAs. Then based on amplicon analysis of every 5 rounds, 18 species of aptamer candidate sequences that have a potential to bind PSTVd were selected. One of them strongly bound and five weakly bound to the circular form of PSTVd RNA in the binding assay, indicated that DNA aptamers specifically bind to PSTVd were successfully selected.
Academic Significance and Societal Importance of the Research Achievements	ウイロイドは約250～430ヌクレオチドの環状1本鎖RNA病原体で、作物の矮化、葉巻、果実障害などの原因になる。近年、野菜・花卉類を中心に、特定重要病害指定のポスピウイロイドが世界中に流行し、国際植物検疫上の重大な懸念材料となっている。国内外の育苗・採種現場ではPCR法などの高感度遺伝子検出法が診断に利用されるが、簡便により効率よく検出する新技術の開発が求められている。本研究で開発したアプタマーはウイロイドの診断・防除薬に利用できるだけでなく、その検出・診断技術を一般化して体系化して公表することは、現在世界中の採種・育苗現場及び植物検疫の最前線で課題となっている問題の解決に貢献するものである。