CONSTRUCTION OF PROTEIN-RESPONSIVE shRNA/RNAi REGULATION SYSTEM UTILIZING RNP MOTIF
| Foreign code | F120006324 |
|---|---|
| File No. | I029P008WO |
| Posted date | Mar 22, 2012 |
| Country | EPO |
| Application number | 09831915 |
| Gazette No. | 2390324 |
| Gazette No. | 2390324 |
| Date of filing | Dec 9, 2009 |
| Gazette Date | Nov 30, 2011 |
| Gazette Date | Oct 1, 2014 |
| International application number | JP2009070580 |
| International publication number | WO2010067811 |
| Date of international filing | Dec 9, 2009 |
| Date of international publication | Jun 17, 2010 |
| Priority data |
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| Title | CONSTRUCTION OF PROTEIN-RESPONSIVE shRNA/RNAi REGULATION SYSTEM UTILIZING RNP MOTIF |
| Abstract |
An object of the present invention is to provide an RNAi control system using an RNA-protein interaction motif. The present invention provides an shRNA comprising: a guide strand having a sequence complementary to a target sequence; a passenger strand which forms a duplex with the guide strand; and a linker strand which links the guide strand and the passenger strand, wherein the linker strand comprises an RNP-derived protein-binding motif sequence. The present invention also provides an RNAi control system comprising: the shRNA; and an RNP-derived protein which specifically binds to a protein-binding motif sequence in the shRNA. |
| Scope of claims |
[claim1] 1. A RNAi control system responsive to a protein expressed in a cell, the system comprising: a vector for expression of a shRNA comprising: a guide strand having a sequence complementary to an mRNA of a target sequence; a passenger strand which forms a duplex with the guide strand; and a linker strand which links the guide strand and the passenger strand, the linker strand comprising a Box CD sequence, wherein the binding of a L7Ae protein or L7Ae protein-containing fusion protein expressed in the cell to the shRNA inhibits the cleavage of the shRNA by Dicer. [claim2] 2. An in vitro RNAi control method responsive to a protein expressed in a cell, the method comprising the step of: introducing into the cell a vector for expression of a shRNA comprising a guide strand having a sequence complementary to an mRNA of a target sequence; a passenger strand which forms a duplex with the guide strand; and a linker strand which links the guide strand and the passenger strand, the linker strand comprising a Box CD sequence, wherein the binding of a L7Ae protein or L7Ae protein-containing fusion protein expressed in the cell to the shRNA inhibits the cleavage of the shRNA by Dicer. [claim3] 3. The RNAi control system according to claim 1, wherein the target sequence of the shRNA is Bcl-xL mRNA, and the RNAi control system controls the expression of an apoptosis regulatory protein. [claim4] 4. A shRNA comprising: a guide strand having a sequence complementary to a mRNA of a target sequence; a passenger strand which forms a duplex with the guide strand; and a linker strand which links the guide strand and the passenger strand, the linker strand comprising a Box CD sequence, wherein in response to a L7Ae protein or L7Ae protein-containing fusion protein expressed in a cell, the cleavage of the shRNA by Dicer is inhibited to control the expression of a protein encoded by the mRNA of the target sequence. [claim5] 5. The RNAi control system according to claim 1, further comprising a vector for intracellular expression of the L7Ae protein or L7Ae protein-containing fusion protein. [claim6] 6. The RNAi control system according to claim 1, wherein the target sequence of the shRNA is GFP mRNA. [claim7] 7. The in vitro RNAi control method according to claim 2, wherein the target sequence of the shRNA is Bcl-xL mRNA, and the RNAi control method controls the expression of an apoptosis regulatory protein. [claim8] 8. The in vitro RNAi control method according to claim 2, further comprising the step of introducing a vector for intracellular expression of the L7Ae protein or L7Ae protein-containing fusion protein into the cell. [claim9] 9. A method for quantifying the expression of an intracellular marker protein comprising an L7Ae protein without destroying a cell, comprising the steps of: introducing a shRNA comprising: a guide strand having a sequence complementary to GFP mRNA; a passenger strand which forms a duplex with the guide strand; and a linker strand which links the guide strand and the passenger strand, the linker strand comprising a Box CD sequence; and measuring the fluorescence intensity of the GFP. |
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| IPC(International Patent Classification) | |
| U.S. Cl./(Sub) |
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| Specified countries | Contracting States: AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO SE SI SK SM TR |
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Contact Information for " CONSTRUCTION OF PROTEIN-RESPONSIVE shRNA/RNAi REGULATION SYSTEM UTILIZING RNP MOTIF "
- Japan Science and Technology Agency Department of Intellectual Property Management
- URL: http://www.jst.go.jp/chizai/
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E-mail:
- Address: 5-3, Yonbancho, Chiyoda-ku, Tokyo, Japan , 102-8666
- Fax: 81-3-5214-8417
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