A Method for Bioluminescence-Based RNA Monitoring Using Split-Luciferase Reconstitution Techniques

Masatoshi Eguchi; Hideaki Yoshimura; Takeaki Ozawa

doi:10.1007/978-1-0716-4248-1_2

A Method for Bioluminescence-Based RNA Monitoring Using Split-Luciferase Reconstitution Techniques

Methods Mol Biol. 2025:2875:9-20. doi: 10.1007/978-1-0716-4248-1_2.

Authors

Masatoshi Eguchi¹, Hideaki Yoshimura¹, Takeaki Ozawa²

Affiliations

¹ Department of Chemistry, School of Science, The University of Tokyo, Tokyo, Japan.
² Department of Chemistry, School of Science, The University of Tokyo, Tokyo, Japan. [email protected].

PMID: 39535636
DOI: 10.1007/978-1-0716-4248-1_2

Abstract

A bioluminescence-based RNA monitoring method in living cells was developed using a split NanoLuc (NLuc) reconstitution technique. For specific recognition of a target RNA sequence, a mutant PUM-HD (mPUM) was used. The method was applied to β-actin mRNA in various cells, including primary cultures of rat hippocampal neurons. It allowed for continuous observation of intracellular localization distribution of the target mRNA in living cells, providing insights into various biological phenomena involving intracellular RNA localization and dynamics.

Keywords: Bioluminescence; Live-cell imaging; Luciferase; RNA; Reconstitution.

MeSH terms

Actins / genetics
Actins / metabolism
Animals
Hippocampus / cytology
Hippocampus / metabolism
Humans
Luciferases / genetics
Luciferases / metabolism
Luminescent Measurements* / methods
Neurons / metabolism
RNA / genetics
RNA, Messenger / genetics
Rats

Substances

Luciferases
Actins
RNA, Messenger
RNA