Small RNA-dependent translational regulatory system in cell or artificial cell model
An object of the present invention is to construct an mRNA which specifically responds to a short RNA sequence and can activate, repress, and regulate the translation of the desired gene, and to construct an artificial cell model system using a liposome comprising the mRNA and a cell-free translational system encapsulated therein. The present invention provides: an mRNA comprising a target RNA-binding site located immediately 5′ to the ribosome-binding site, and a nucleotide sequence located 5′ to the target RNA-binding site, the nucleotide sequence being complementary to the ribosome-binding site; an mRNA comprising a small RNA-binding site located 3′ to the start codon, and a nucleotide sequence located 3′ to the small RNA-binding site, the nucleotide sequence encoding a protein; and a liposome comprising any of these mRNAs encapsulated therein.
1. An mRNA comprising a microRNA-binding site located 5′ to the ribosome-binding site and a nucleotide sequence located 5′ to the microRNA-binding site, the nucleotide sequence being complementary to the ribosome-binding site.
2. The mRNA according to claim 1 , wherein the microRNA-binding site has a sequence complementary to a microRNA selected from miR164, miR170, miR171, miR156, miR159a or miR163.
3. A liposome comprising an mRNA according to claim 1 encapsulated therein.
4. The liposome according to claim 3 , wherein the liposome is produced by a method comprising steps of:
mixing one or more phospholipids, the mRNA, a cell-free translation system, and an aqueous solution into an oily liquid to form a W/O emulsion in which the mRNA and the cell-free translational system are encapsulated in the phospholipid vesicle;
adding an oily liquid containing outer membrane lipids dissolved therein, to an aqueous phase to form a molecular membrane in which the lipids are arranged at the oil/water interface; and
adding the W/O emulsion to the oil phase side of the interface and moving the W/O emulsion to the aqueous phase side of the interface such that the outer membrane lipid is added outside of the W/O emulsion to form a liposome.
5. A translation/expression regulation system comprising an mRNA according to claim 1 .
6. The translation/expression regulation system according to claim 5 , further comprising a microRNA complementary to the microRNA-binding site.