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COMMON HIGH-SPEED PHOTO-CROSS-LINKING LINKER FOR MOLECULAR INTERACTION ANALYSIS AND IN VITRO SELECTION, AND IN VITRO SELECTION METHOD USING LINKER

外国特許コード F160008912
整理番号 (S2015-0929-N0)
掲載日 2016年12月6日
出願国 世界知的所有権機関(WIPO)
国際出願番号 2016JP060611
国際公開番号 WO 2016159211
国際出願日 平成28年3月31日(2016.3.31)
国際公開日 平成28年10月6日(2016.10.6)
優先権データ
  • 特願2015-072810 (2015.3.31) JP
発明の名称 (英語) COMMON HIGH-SPEED PHOTO-CROSS-LINKING LINKER FOR MOLECULAR INTERACTION ANALYSIS AND IN VITRO SELECTION, AND IN VITRO SELECTION METHOD USING LINKER
発明の概要(英語) The purpose of the present invention is to provide a linker that can be used for both candidate clone screening and assessment of the binding of the resulting candidate clones, without using enzymes, and to provide an in vitro selection method using the linker. The present invention provides a common high-speed photo-cross-linking linker for molecular interaction analysis and in vitro selection comprising a main chain and a side chain. The main chain has: a solid-phase binding site located at the 5' terminus for forming a bond with a solid-phase; solid-phase cleavage site for separating the solid-phase by separating the entire solid-phase binding site; a side chain linking site for linking a side chain; a high-speed photo-cross-linking site for linking the main chain to mRNA having a sequence complementary to the main chain by photo-cross-linking; and a reverse transcription initiation region located adjacent to the side chain linking site at the 3' terminus of the main chain. The side chain has: a fluorescent label; a protein binding site located at the free terminus; and a link forming site that links to the side chain linking site on the main chain.
特許請求の範囲(英語) [claim1]
1. Being the linker which possesses with the principal chain and the side chain,
As for the aforementioned principal chain,
The solid phase binding site in order it possesses specified base arrangement, is categorized 5' to the end of the aforementioned principal chain and to form the connection with the solid phase;
Every aforementioned solid phase binding site the solid phase cutting region in order to separate the aforementioned solid phase;
The side chain connected region in order to connect the aforementioned side chain;
The high-speed optical building a bridge region which is located with the aforementioned side chain connected region and the aforementioned solid phase cutting region, it connects aforementioned principal chain and mRNA which possesses complimentary arrangement, with aforementioned principal chain and optical building a bridge;
The reversal copying start territory which it adjoins to the aforementioned side chain connected region, is categorized 3' to the end of the aforementioned principal chain;
Having,
As for the aforementioned side chain, the fluorescent sign and the protein binding site which is categorized to separation end and the connected formation region which is connected to the side chain connected region of the aforementioned principal chain having,
The aforementioned side chain is connected to the aforementioned side chain connected region with the aforementioned connected formation region, high-speed optical building a bridge type common linker for interaction analysis during test inside of pipe selecting and the molecule.
[claim2]
2. In the claim 1 where the aforementioned solid phase cutting region, [deokishiinoshin], ribo- G or is formed with the base which is chosen from the group which consists of ribo- pyrimidine features thing high-speed optical building a bridge type common linker for interaction analysis during test inside of pipe selecting and the molecule of statement.
[claim3]
3. In the claim 1 where the aforementioned high-speed optical building a bridge region is formed with the cyano vinyl carbazole compound, features thing or 2 high-speed optical building a bridge type common linker for interaction analysis during test inside of pipe selecting and the molecule of statement.
[claim4]
4. The aforementioned cyano vinyl carbazole compound 3 - is cyano vinyl carbazole, in the claim 3 which features thing high-speed optical building a bridge type common linker for interaction analysis during test inside of pipe selecting and the molecule of statement.
[claim5]
5. The aforementioned solid phase binding site, the biotin, [sutoreputoabijin], alkyne and in either of the claim 1-4 where the agitation chemical material by click chemistry, the amino group, [N]-hidorokishisukushinimidoesuteru (NHS), with poly- A which is connected to each chemical compound and the aforementioned chemical compound which are chosen from the SH basis, and the group which consists of Au is formed, thing features high-speed optical building a bridge type common linker for interaction analysis during test inside of pipe selecting and the molecule of statement.
[claim6]
6. In either of the claim 1-5 where the aforementioned protein binding site the puromycin or is formed with that affinity chemical compound, features thing high-speed optical building a bridge type common linker for interaction analysis during test inside of pipe selecting and the molecule of statement.
[claim7]
7. The affinity chemical compound of the aforementioned puromycin 3' - the [N]-amino acyl puromycin and 3' - is each chemical compound which is chosen from the group which consists of the nucleoside of [N]-amino acyl adenosine amino acid, in the claim 6 which features thing high-speed optical building a bridge type common linker for interaction analysis during test inside of pipe selecting and the molecule of statement.
[claim8]
8. In claim 1 the complimentary connection formation process which principal chain of high-speed optical building a bridge type common linker for interaction analysis during test inside of pipe selecting and the molecule of statement and mRNA of desire compliment is made to connect;
Between 0.5-5 amount irradiating the light of the wave length of 300-400nm with to principal chain and mRNA where the aforementioned complimentary connection was formed, the optical building a bridge process which makes optical building a bridge form;
MRNA which is connected to the aforementioned linker is translated with non cell translation system, the protein which corresponds to the description above mRNA is made to connect to the protein binding site of the aforementioned linker, the fusion body formation process which the fusion body of [mRNA]-tanpaku is formed;
The solid phase connection process which makes the aforementioned fusion body the solid phase connect;
MRNA which is included in the aforementioned fusion body the reversal copy, the aforementioned fusion body and reversal the reversal copying process which forms the joining union cDNA which is copied with;
The selective process which cuts off the aforementioned joining union from the solid phase cutting region, selects the cDNA display method of desire;
It has, test inside of pipe selection method.
[claim9]
9. The aforementioned solid phase is the magnetic particle which coating is done with [sutoreputoabijin] or [abijin], in the claim 8 which features thing test inside of pipe selection method of statement.
[claim10]
10. In the claim 8 which cuts off the aforementioned joining union in the aforementioned selective process, endonuclease V, features thing with each enzyme which is chosen from the group which consists of RNase T1, and RNase A or 9 test inside of pipe selection method of statement.
[claim11]
11. The principal chain of the aforementioned high-speed optical building a bridge type common linker has the arrangement which recognizes the sugar chain antigen, thing is featured, in either of the claim 8-10 test inside of pipe selection method of statement.
[claim12]
12. In claim 1 the complimentary connection formation process which principal chain of high-speed optical building a bridge type common linker for interaction analysis during test inside of pipe selecting and the molecule of statement and mRNA of desire compliment is made to connect;
Between 0.5-5 amount irradiating the light of the wave length of 300-400nm with to principal chain and mRNA where the aforementioned complimentary connection was formed, the optical building a bridge process which makes optical building a bridge form;
MRNA which is connected to the aforementioned linker is translated with non cell translation system, the protein which corresponds to the description above mRNA is made to connect to the protein binding site of the aforementioned linker, the fusion body formation process which the fusion body of [mRNA]-tanpaku is formed;
Digesting the aforementioned fusion body with RNA, linker - it makes the protein fusion body, linker - protein fusion body formation process;
The aforementioned linker - the solid phase connection process which makes the protein fusion body the solid phase connect;
The refinement process which the aforementioned linker - under specified condition makes the protein fusion body liquate from the aforementioned solid phase, refines;
The linker for affinity measurement which it has - production method of the protein.
[claim13]
13. The aforementioned solid phase is the magnetic particle which coating is done with [sutoreputoabijin] or [abijin], in the claim 12 which features thing linker for affinity measurement of statement - production method of the protein.
[claim14]
14. In the claim 12 which in the aqueous solution which includes 0-100mM NaCl, at room temperature does the aforementioned refinement process, features thing or 13 linker for affinity measurement of statement - production method of the protein.
[claim15]
15. Is produced with method of statement the linker for affinity measurement which - the protein either of the claim 12-14.
  • 出願人(英語)
  • ※2012年7月以前掲載分については米国以外のすべての指定国
  • SAITAMA UNIVERSITY
  • 発明者(英語)
  • NEMOTO NAOTO
  • MOCHIZUKI YUKI
  • KUMACHI SHIGEFUMI
国際特許分類(IPC)
指定国 (WO2016159211)
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 DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IR IS JP KE KG KN KP KR 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 SM ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN 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
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