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MODIFIED Α-HEMOLYSIN AND NANOPORE SEQUENCER USING SAME

Foreign code F150008482
File No. E102P06WO
Posted date Oct 26, 2015
Country WIPO
International application number 2014JP069540
International publication number WO 2015016125
Date of international filing Jul 24, 2014
Date of international publication Feb 5, 2015
Priority data
  • P2013-157799 (Jul 30, 2013) JP
Title MODIFIED Α-HEMOLYSIN AND NANOPORE SEQUENCER USING SAME
Abstract Provided is a modified α-hemolysin in which a mutation is introduced to the amino acid sequence of an α-hemolysin monomer and which has arginine at the 147th position or glutamine at the 146th position from the N terminal of the amino acid sequence, and due to this modified α-hemolysin, the ability to form a nanopore in a lipid membrane is enhanced without reducing enzyme activity when compared to the wild form.
Outline of related art and contending technology BACKGROUND ART
Α - hemolysin is, secreted from Staphylococcus aureus 7 (Staphylococcus aureus) the amount of the poison protein, the protein is water-soluble, open nanopores in the lipid bilayer membrane, red blood cells to activity. Of a material having a molecular weight of about 3kDa in a bi-directional diffusion (transmission) can be synthesized using a function of promoting biological used in the field. Α - hemolysin is, the amount of the 7 for the first time, the lipid film to form pores (nanopores) can be known.
Α - using such toxins, have been proposed one after another generation sequencer (for example, in Japanese Unexamined Patent Publication 2011-527191 (Patent Document 1)). That is, the amount of 7 α - in the lipid bilayer membrane made of a hemolysin nanopore is formed, through which the chain DNA sequence information technology. That is, a voltage is applied across the nanopore and the ion current flows, and pass through pores of the nano-chain DNA at the moment of change in the current value detected for each base 1, to obtain sequence information. At this time, the kind of the base 4 so as to improve the distinguishing DNA sequences can result can be obtained. Compared with the conventional sequencer, and is easy to adjust the sample, such as on the fragmentation processing is unnecessary, and to some extent in the blood dirty solution can be used to directly analyze DNA. And the nucleotide sequence in real time, the nucleotide sequence obtained by analyzing the user an intended to obtain the result sequencing can be continued, because of their advantages.
However, in current technology is the limited number of nanopore chip 1 (approximately 10000) and, as an array of numbers read unit billion to decrypt the nano pore formation and to improve the efficiency of which require some contrivance such as (for example, protein translocation through an α-hemolysin nanopore J. Nivala et al., "Unfoldase-mediated", Nature Biotechnology,2013,31 (3), (non-patent document 1) pp.247-250 reference.).
From this point of view, the ability of the lipid membrane higher than the nano-pore-forming toxins which is where it is desirable to provide α -.
The past, a hemolysin for α -, enzyme due to the introduction of point mutations have been attempted modification technique is, causes a reduction of the enzymatic activity is in most cases (for example, b. Walker et al., "Key Residues for Membrane Binding, Oligomerization, and Pore Forming Activity of Staphylococcal α-Hemolysin Identified by Cysteine Scanning Mutagenesis and Targeted Chemical Modification", The Journal of Biological Chemistry,1995,Vol.270,No.39,pp.23065-23071( non-patent document 2) reference.).
Scope of claims (In Japanese)請求の範囲 [請求項1]
 α-ヘモリシン単量体のアミノ酸配列に変異が導入された改良型のα-ヘモリシンであって、
 前記アミノ酸配列のN末端側から147番目の位置にアルギニンを有するか、または、146番目の位置にグルタミンを有する、改良型α-ヘモリシン。

[請求項2]
 前記アミノ酸配列のN末端側から122番目の位置にセリンもしくはアスパラギン酸を有するか、または、121番目の位置にイソロイシンを有する、請求項1に記載の改良型α-ヘモリシン。

[請求項3]
 前記アミノ酸配列のN末端側から147番目の位置にアルギニンを有し、かつ、122位にセリンを有する、請求項2に記載の改良型α-ヘモリシン。

[請求項4]
 前記アミノ酸配列のN末端側から147番目の位置にアルギニンを有し、かつ、121番目の位置にイソロイシンを有する、請求項2に記載の改良型α-ヘモリシン。

[請求項5]
 前記アミノ酸配列のN末端側から147番目の位置にアルギニンを有し、かつ、122番目の位置にアスパラギン酸を有する、請求項2に記載の改良型α-ヘモリシン。

[請求項6]
 配列番号4、6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36、38または40のアミノ酸配列を有する、請求項1に記載の改良型α-ヘモリシン。

[請求項7]
 請求項1~6のいずれか1項に記載の改良型α-ヘモリシンが7個集積して形成された7量体と、当該7量体が埋め込まれた脂質膜とを有する基板を備え、当該7量体により脂質膜を貫通するナノポアが形成された、ナノポアシーケンサ。

[請求項8]
 脂質膜を構成する脂質がホスファチジルコリンを含む、請求項7に記載のナノポアシーケンサ。

[請求項9]
 請求項7に記載のナノポアシーケンサに用いるための基板であって、
 前記改良型α-ヘモリシンが7個集積して形成された7量体と、当該7量体が埋め込まれた脂質膜とを備える、基板。

  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • JAPAN SCIENCE AND TECHNOLOGY AGENCY
  • Inventor
  • YOMO, Tetsuya
  • MATSUURA, Tomoaki
  • KAZUTA, Yasuaki
  • FUJII, Satoshi
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 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 LT 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 TD TG
Reference ( R and D project ) ERATO YOMO Dynamical Micro-scale Reaction Environment AREA
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