Construction and application of a built-in dual luciferase reporter for microRNA functional analysis

Yanzhen Bi*¹ · Xinmin Zheng¹ · Changwei Shao² · Wen Pan³ · Li Jiang² · Huiwu Ouyang²

¹Hubei Key Laboratory of Animal Embryo Engineering and Molecular Breeding, Institute of Animal Husbandry and Veterinary, Hubei Academy of Agricultural Science, China
²RNA Group, College of Life Science, Wuhan University, China
³The Third Affiliated Hospital of Sun Yat-Sen University, Tianhe District, China

*Corresponding author: biyanzhen@gmail.com

Financial support: This work was funded by a research grant from Hubei Key Laboratory of Animal Embryo Engineering and Molecular Breeding to Y. Z. Bi (2010ZD163).

Keywords: biosensor, luciferase, ligase-independent, miRNA, target.

Background: As key gene regulators, microRNAs post-transcriptionally modulate gene expression via binding to partially complementary sequence in the 3’ UTR of target mRNA. An accurate, rapid and quantitative tool for sensing and validation of miRNA targets is of crucial significance to decipher the functional implications of miRNAs in cellular pathways.
Results: Taking advantage of an improved restriction-free cloning method, we engineered a novel built-in dual luciferase reporter plasmid where Firefly and Renilla luciferase genes were assembled in a single plasmid named “pFila”. This design eliminates the transfection of a separate control plasmid and thus minimizes the time and labor required for miRNA-target sensing assays. pFila consistently produces Firefly and Renilla luciferase activities when transfected into human-, monkey- and mouse-derived mammalian cell systems. Moreover, pFila is capable of recapitulating the interaction of miR-16 and its known target CCNE1 in Hela cells. Additionally, pFila is shown to be a sensitive miR-biosensor by evaluating the inhibition efficiency of endogenous miRNA.
Conclusions: pFila would facilitate miRNA target identification and verification in a rapid and simplified manner. Also, pFila is a sensitive biosensor for active miRNA profiling in vivo.