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NUCLEIC ACID BINDING PROTEIN

Foreign code F210010309
File No. AF46-03WO
Posted date Jan 29, 2021
Country WIPO
International application number 2020JP015966
International publication number WO 2020209332
Date of international filing Apr 9, 2020
Date of international publication Oct 15, 2020
Priority data
  • P2019-074004 (Apr 9, 2019) JP
Title NUCLEIC ACID BINDING PROTEIN
Abstract The present invention provides a novel modified protein which is to be used, as a novel detection tool relating to gene expression, for detecting a chromatin open structure more easily at a higher sensitivity than by the conventional technique. The present invention relates to: a nucleic acid binding fluorescent protein, said protein containing a DNA binding domain in which 3 or more TAL-repeats are repeatedly connected, characterized by binding independently from base sequences; and a method for fluorescent labeling of an open chromatin in a vital cell, said method comprising a step for transferring a gene encoding a nucleic acid binding protein into the vital cell, characterized in that the nucleic acid binding protein is a protein comprising a DNA binding domain, in which 3 or more TAL-repeats are repeatedly connected, and a fluorescent protein directly or indirectly bound thereto and the DNA binding domain binds to a nucleic acid independently from base sequences.
Outline of related art and contending technology BACKGROUND ART
In recent years, attention has been paid to the results of the study on the mechanism of diversity of individual cells in spite of the same genome information. From the progress of this study, it was found that the maintenance of the essential genes and the suppression of the unnecessary genes are correctly regulated. The cell-specific trait appearing as a result of such control is maintained stably beyond cell division, and this event is called cell memory (or epigenetic).
TAL (Transcription Activator-Like) Effector (TALE) was identified as a transcription factor involved in gene expression regulation to suppress the immune mechanism of host cells when infected with pathogenic bacteria. TALE includes, at its center, a DNA binding domain consisting of a portion in which a structural unit (module) consisting of about 34 amino acids called TAL-repeat is linked by 10 ~ 30 repetitions. TAL-repeat consists of 2 set structures, where there are 2 amino acid residues involved in nucleobase recognition called Repeat Variable Diresidue (RVD) in the loop region connecting the 2 set structures. Individual TAL-repeat recognizes 1 nucleobases depending on the amino acid sequence of each RVD.
TALE can be modified so as to selectively bind to a nucleic acid containing a specific base sequence by linking TAL-repeat having RVD specifically recognizing a specific nucleic acid base in an appropriate order. That is, sequence-dependent binding can be made possible by using a module corresponding to the DNA sequence targeted for the DNA binding domain of TALE. For example, in genome editing, TALEN (TALE-Nuclease) in which TALE and FokI nuclease which recognize in dependence on the nucleic acid sequence are fused is used.
On the other hand, organisms have been known to have open chromatin structures by partially loosening aggregates of nucleic acids and histone in the nucleus in the regulation of expression of various genes in the development and differentiation. Analysis of whether or not the open chromatin structure takes place is particularly important in functional analysis of unidentified genes such as expression and identification of unknown differentiation-inducing genes involved in specific diseases, expression and identification of unidentified genes involved in carcinogenesis, and expression and identification of genes involved in metabolic abnormalities and neurodegeneration based on regulatory mechanisms which have not been elucidated yet.
Scope of claims (In Japanese)[請求項1]
 3個以上のTAL-repeatが繰り返し連結されたDNA結合ドメインを含有し、塩基配列非依存的に核酸に結合することを特徴とする、核酸結合性タンパク質。

[請求項2]
 前記TAL-repeatのRVD配列が、HT、RS、又はHSである、請求項1に記載の核酸結合性タンパク質。

[請求項3]
 前記DNA結合ドメインにおける前記TAL-repeatの繰り返し数が、3個以上35個未満である、請求項1又は2に記載の核酸結合性タンパク質。

[請求項4]
 前記DNA結合ドメインは、同一アミノ酸配列からなる前記TAL-repeatが繰り返し連結されている、請求項1~3のいずれか一項に記載の核酸結合性タンパク質。

[請求項5]
 前記DNA結合ドメインが、前記TAL-repeatのRVD配列がHT、RS、又はHSであるように改変されたTALEのDNA結合ドメインである、請求項1~4のいずれか一項に記載の核酸結合性タンパク質。

[請求項6]
 前記DNA結合ドメインに含まれている前記TAL-repeatは、キサントモナス属細菌由来のTALEのTAL-repeat、又は前記TAL-repeatのRVD以外の領域にアミノ酸変異が導入されているTAL-repeatである、請求項1~5のいずれか一項に記載の核酸結合性タンパク質。

[請求項7]
 前記DNA結合ドメインを含有するタンパク質と蛍光タンパク質とが、直接又は間接的に結合したタンパク質である、請求項1~6のいずれか一項に記載の核酸結合性タンパク質。

[請求項8]
 請求項1~7のいずれか一項に記載の核酸結合性タンパク質からなり、オープンクロマチン構造と選択的に結合する、タンパク質プローブ。

[請求項9]
 請求項1~7のいずれか一項に記載の核酸結合性タンパク質をコードする塩基配列を含有する、核酸分子。

[請求項10]
 請求項9に記載の核酸分子が組込まれており、宿主細胞において、請求項1~7のいずれか一項に記載の核酸結合性タンパク質を発現し得る、発現ベクター。

[請求項11]
 生細胞に、核酸結合性タンパク質をコードする遺伝子を導入する工程を有し、
 前記核酸結合性タンパク質が、3個以上のTAL-repeatが繰り返し連結されたDNA結合ドメインと蛍光タンパク質とを含むタンパク質であり、
 前記DNA結合ドメインが、塩基配列非依存的に核酸と結合することを特徴とする、生細胞中のオープンクロマチンを蛍光標識する方法。

[請求項12]
 請求項1~7のいずれか一項に記載の核酸結合性タンパク質をコードする遺伝子が導入された、形質転換細胞(但し、ヒトを構成する細胞を除く)。

[請求項13]
 請求項1~7のいずれか一項に記載の核酸結合性タンパク質をコードする遺伝子が導入された、形質転換非ヒト動物。
  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • JAPAN SCIENCE AND TECHNOLOGY AGENCY
  • Inventor
  • OKADA Yasushi
  • INO Daisuke
  • IKEDA Kazuho
IPC(International Patent Classification)
Specified countries National States: AE AG AL AM AO AT AU AZ BA BB BG BH BN BR BW BY BZ CA CH CL CN CO CR CU CZ DE DJ DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IR IS JO JP KE KG KH KN KP KR KW KZ LA LC LK LR LS LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PA PE PG PH PL PT QA RO RS RU RW SA SC SD SE SG SK SL ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN WS ZA ZM ZW
ARIPO: BW GH GM KE LR LS MW MZ NA RW SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ RU TJ TM
EPO: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW KM ML MR NE SN ST TD TG
Reference ( R and D project ) CREST Innovative Technology Platforms for Integrated Single Cell Analysis AREA
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