Protein-responsive translational regulatory system using RNA-protein interacting motif

Patent metadata

Field	Value
Publication number	US-9255274-B2
Application number	US-74385308-A
Country	US
Kind code	B2
Filing date	Nov 21, 2008
Priority date	Nov 22, 2007
Publication date	Feb 9, 2016
Grant date	Feb 9, 2016

Abstract

An object of the present invention is to provide a translationally regulatable mRNA which has wider application and can perform specific ON-OFF regulation, an RNA-protein complex specifically bound to the mRNA, and a translational regulatory system. The present invention provides an mRNA having an RNA-protein complex interacting motif-derived nucleotide sequence 5′ to the ribosome-binding site or within the 5′ region of the open reading frame, and an mRNA having a nucleotide sequence complementary to an RNA-protein complex interacting motif-derived nucleotide sequence 5′ to the ribosome-binding site or within the 5′ region of the open reading frame.

First claim

The invention claimed is:

1. An isolated non-naturally occurring mRNA encoding a protein comprising an RNA-protein complex interacting motif nucleotide sequence incorporated 5′ to a ribosome-binding site in a position 2 to 10 bases distant from the ribosome-binding site or within the 5′ region of an open reading frame, wherein the interacting motif comprises nucleic acid sequence SEQ ID NO:9, wherein an RNA-protein complex of the interacting motif and L7Ae protein has a dissociation constant Kd of approximately 0.1 nM to approximately 1 μM between the motif and L7Ae and the interacting motif interacts with L7Ae protein.

2. An RNA-protein complex comprising an mRNA according to claim 1 and a protein specifically binding to the nucleotide sequence.

3. A translational regulatory kit comprising an mRNA according to claim 1 and a protein specifically binding to the nucleotide sequence.

4. A method for translational regulation of mRNA, comprising contacting the mRNA according to claim 1 with a protein specifically binding to the RNA-protein complex interacting motif nucleotide sequence.

5. An artificial information conversion method which converts input information of an arbitrary substrate protein to output information of an arbitrary target protein using an mRNA according to claim 1 , comprising steps of
preparing the mRNA of claim 1 having an open reading frame encoding the arbitrary target protein; and
contacting the mRNA with the substrate protein that specifically binds to the RNA-protein complex interacting motif nucleotide sequence.

6. A plasmid vector comprising a nucleic acid sequence encoding an mRNA according to claim 1 .

7. An intracellular translational regulatory kit comprising
a first plasmid vector comprising a nucleic acid sequence encoding an mRNA according to claim 1 , and
a second plasmid vector comprising a nucleic acid sequence encoding a protein specifically binding to the RNA-protein complex interacting motif nucleotide sequence.

8. The kit according to claim 7 , for regulating protein translation in a human cancer cell.

Assignees

• Inoue Tan
• Saito Hirohide
• Kobayashi Tetsuhiro
• Hara Tomoaki
• Japan Science & Tech Agency

Inventors

• Inoue Tan
• Saito Hirohide
• Kobayashi Tetsuhiro
• Hara Tomoaki

Classifications

• C12N15/67 Primary

  General methods for enhancing the expression · CPC title