Development of opto-bioanalysis to measure the amount and activity of biomolecules

2022 Fiscal Year Final Research Report

• Project/Area Number: 19H00900
• Research Category: Grant-in-Aid for Scientific Research (A)
• Allocation Type: Single-year Grants
• Section: 一般
• Review Section: Medium-sized Section 34:Inorganic/coordination chemistry, analytical chemistry, and related fields
• Research Institution: The University of Tokyo
• Principal Investigator: Ozawa Takeaki 東京大学, 大学院理学系研究科(理学部), 教授 (40302806)
• Co-Investigator(Kenkyū-buntansha): 尾崎 倫孝 北海道大学, 保健科学研究院, 教授 (80256510)
• Project Period (FY): 2019-04-01 – 2022-03-31
• Keywords: 光操作 / RNA / 酵素 / 標準添加法

Outline of Final Research Achievements

The research objective was to develop an innovative technique for quantifying RNA and enzyme activity in a specific single cell. In the first project, we developed a standard addition method for quantifying enzyme activity using photo-inactivated luciferase (PI-Luc). PI-Luc was synthesized in large quantities using an Escherichia coli expression system, and the purified product was used to measure luminescent activity under blue light irradiation. To suppress phototoxicity, we established a light-controlling system using near-infrared light with upconversion nano particles. In the second project, we developed RNA luminescence probes with luciferase-fragment complementation and succeeded in bioluminescence detection of β-actin mRNA and telomere RNA. These bioluminescent probes have a wide dynamic range of detection and are excellent for quantitative analysis.

Free Research Field

分析化学

Academic Significance and Societal Importance of the Research Achievements

生細胞内の特定分子や酵素を可視化するためのプローブは，蛍光プローブをはじめ様々開発され，その細胞内の時空間ダイナミクスを解明する重要な基盤分析技術となった．一方，蛍光プローブには定量性に問題があり大きな課題となっている．本研究成果は，応答のダイナミックレンジが広いルシフェラーゼの発光検出の利点を活かすことで，細胞内のRNAや酵素活性を定量しかつ時空間動態を解析できる分析法を新たに創出した点に分析化学の学術的インパクトがある．基礎生物学研究や細胞センサーの開発など広範な応用展開が期待され，検出機器の進歩とともに今後の大きな発展が見込まれる技術である．