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

外国特許コード F110003837
整理番号 I029P004WO
掲載日 2011年7月6日
出願国 アメリカ合衆国
出願番号 74385308
公報番号 20110040077
公報番号 9255274
出願日 平成20年11月21日(2008.11.21)
公報発行日 平成23年2月17日(2011.2.17)
公報発行日 平成28年2月9日(2016.2.9)
国際出願番号 JP2008071213
国際公開番号 WO2009066757
国際出願日 平成20年11月21日(2008.11.21)
国際公開日 平成21年5月28日(2009.5.28)
優先権データ
  • 特願2007-303662 (2007.11.22) JP
  • 特願2008-186385 (2008.7.17) JP
  • 2008JP071213 (2008.11.21) WO
発明の名称 (英語) Protein-responsive translational regulatory system using RNA-protein interacting motif
発明の概要(英語) 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.
従来技術、競合技術の概要(英語) BACKGROUND ART
With the progress of post-genomic science, information has accumulated about the structures and functions of biomolecules such as proteins or RNAs.
There has been a growing tendency of synthetic biology, which exploits such increasing information to understand the systems of life through "synthesis", in contrast to previous reductive or analytical biology.
Particularly, the artificial (re)construction of biomolecules or genetic circuits has received considerable attention in terms of not only life science research but also industrial application.
Particularly, there has been a demand for the progress of translational regulatory systems which can recognize a particular protein and regulate arbitrary gene expression.
Heretofore, the conventional technique is known, in which the induction of transcription of DNA is regulated by small molecules or proteins (see Non-Patent Document 1).
This technique is a method for modulating the regulation of transcription from DNAs to RNAs.
However, this technique had the problem that it cannot be applied directly as a technique of regulating translation from RNAs to proteins.
Moreover, there is a naturally occurring system (S15, ThrRS, etc.) in which the protein regulates a translation level upon binding to its own mRNA 5' untranslated region (5'-UTR).
However, no artificial translational repression/activation system of a target gene using such an RNP interacting motif has been constructed intracellularly or extracellularly.
Moreover, RNAs called "riboswitches", in which mRNAs induce structural change in response to metabolites, resulting in the regulation of gene expression, have been discovered in recent years in bacteria and have received attention.
However, natural riboswitches use substrates limited to small molecules such as vitamins or amino acids and therefore, cannot regulate gene expression in response to biomacromolecules such as RNAs or proteins.
Furthermore, natural riboswitches are limited to systems for performing the feedback regulation of their own expressions and therefore, have not been applied so far to the development of artificial systems that regulate arbitrary gene expression.
Thus, the development of artificial riboswitches having such functions has been expected.
The conventional technique is known as to translational regulation using RNA aptamers or antisense.
There also exists a technique which involves introducing a small molecule theophylline-binding aptamer into an artificial RNA using yeast to prepare an "RNA switch" which performs ON/OFF regulation of gene expression in a manner dependent on the presence of theophylline (Non-Patent Document 2).
However, this technique had the problem that it is a system responding to the aptamer for small molecules and therefore, cannot be applied to biomacromolecules such as proteins as substrates.
Non-Patent Document 1: Trends Biochem Sci. 2005; 30 (6): 275-9
Non-Patent Document 2: Nat Biotechnol. 2004 22 (7): 841-7. 2004

特許請求の範囲(英語) [claim1]
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 mu M between the motif and L7Ae and the interacting motif interacts with L7Ae protein.
[claim2]
2. An RNA-protein complex comprising an mRNA according to claim 1 and a protein specifically binding to the nucleotide sequence.
[claim3]
3. A translational regulatory kit comprising an mRNA according to claim 1 and a protein specifically binding to the nucleotide sequence.
[claim4]
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.
[claim5]
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.
[claim6]
6. A plasmid vector comprising a nucleic acid sequence encoding an mRNA according to claim 1.
[claim7]
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.
[claim8]
8. The kit according to claim 7, for regulating protein translation in a human cancer cell.
  • 発明者/出願人(英語)
  • INOUE TAN
  • SAITO HIROHIDE
  • KOBAYASHI TETSUHIRO
  • HARA TOMOAKI
  • JAPAN SCIENCE AND TECHNOLOGY AGENCY
国際特許分類(IPC)
米国特許分類/主・副
  • 530/409
  • 435/320.1
  • 530/350
  • 536/23.1
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