Visualization of the intracellular dynamics of artificial mRNAs

2021 Fiscal Year Final Research Report

• Project/Area Number: 20K22704
• Research Category: Grant-in-Aid for Research Activity Start-up
• Allocation Type: Multi-year Fund
• Review Section: 0801:Pharmaceutical sciences and related fields
• Research Institution: Nagoya University
• Principal Investigator: Hiraoka Haruka 名古屋大学, 理学研究科, 研究員 (70880053)
• Project Period (FY): 2020-09-11 – 2022-03-31
• Keywords: 化学修飾核酸 / RNAイメージング / 翻訳 / 細胞デリバリー

Outline of Final Research Achievements

In this research theme, I tried to develop the RNA imaging method using fluorescence nucleic acid probe. The nucleic acid probe used for bacteria in previous research could not be used in human cells as such short oligonucleotide easily permeated into nuclei and rarely bound with target mRNA in cytosol. Therefore, instead of using the nucleic acid probe, I currently deal with the development of fluorescence mRNA composed with fluorescence nucleotides. I also achieved the intracellular delivery of the nucleic acid probe by chemical modification. Previously developed membrane-permeable oligonucleotide including 5 repeats of disulfide modification units has limited use due to their high hydrophobicity. This study reported circular disulfide group as promising modification; it enables the efficient intracellular delivery with smaller number of units.

Free Research Field

細胞生物学・分子生物学・核酸化学

Academic Significance and Societal Importance of the Research Achievements

RNAイメージングに用いる蛍光核酸プローブをダメージなく細胞に導入するために最適な化学修飾の探索を行った結果、環状ジスルフィド修飾を付加することで細胞毒性なく効果的に細胞導入できることが分かった。従来の膜透過性核酸よりも少ない修飾ユニット数で細胞膜透過を実現しており、疎水性も遥かに低い。その優れた物性から、蛍光前駆体など他の修飾基との併用や、1000塩基を超える長いmRNAの導入への適用など高い汎用性が期待される。また、本手法はエンドサイトーシス等を介さず直接的に細胞質へと導入できる。この技術を用いて、蛍光mRNAの導入から翻訳に至る過程を経時的に観察することを目指す。