Top > Search of International Patents > COMMON HIGH-SPEED PHOTO-CROSS-LINKING LINKER FOR MOLECULAR INTERACTION ANALYSIS AND IN VITRO SELECTION, AND IN VITRO SELECTION METHOD USING LINKER

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

Foreign code F160008912
File No. (S2015-0929-N0)
Posted date Dec 6, 2016
Country WIPO
International application number 2016JP060611
International publication number WO 2016159211
Date of international filing Mar 31, 2016
Date of international publication Oct 6, 2016
Priority data
  • P2015-072810 (Mar 31, 2015) JP
Title COMMON HIGH-SPEED PHOTO-CROSS-LINKING LINKER FOR MOLECULAR INTERACTION ANALYSIS AND IN VITRO SELECTION, AND IN VITRO SELECTION METHOD USING LINKER
Abstract 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.
Outline of related art and contending technology BACKGROUND ART
Up to now, the development of antibody pharmaceuticals by each pharmaceutical company have been performed. Antibody, placed for a certain period of antigen molecules is administered to a plurality of times, then, the blood must be purified from a blood, in the in vivo production of the amount of time and effort. In addition, even the smallest molecular weight IgG 150 KDa as large molecular weight, difficult to synthesize a completely in vitro.
On the other hand, the antibody or the antigen recognition site of the peptide aptamers, small molecular weight can be artificially synthesized.
In the case of use as a pharmaceutical GMP level is found in organic synthesis, peptide aptamers as described above in the case of the synthesis of these levels are also possible. In addition, the molecular weight is small can be easy and the chemically-modified, immobilized on the chip or the like can be freely and, further, high stability so compared to the antibody, were fixed to the chip or the like, can be stored at room temperature such advantages as the.
Such peptide aptamers, after creating mRNA, techniques associated with the genotype - phenotype (hereinafter, simply referred to as' association techniques' may also be referred to.) Can be synthesized using and selection. Then, in association with such techniques include, outside the display methods cDNA, phage display, ribosome display method, mRNA display methods and the like are present. Of these, automated, high throughput in consideration of the request, to synthesize cDNA display method is most appropriately on the basis of the peptide aptamers.
Conventional, cDNA for use in display methods, as shown in Fig. 1A - 1D proposed linker, have been used. 1A In the FIG., solid phase binding sites and, with the main chain of the linker and the mRNA T4 RNA ligase for T4 RNA ligase so as to couple the connection portion, the base region and a primer for reverse transcription of the main chain, a peptide binding site and fluorescent labels, the main chain is connected to the side - chain linker is shown (see Patent Document 1, or less, '1 conventional example' referred to.).
In addition, Fig. 1B is, the double-stranded part of the main chain is composed of a conventional example 1 except that the linker has substantially the same structure, composed of the double-stranded portion, to separate the linker from the solid phase linker incorporated restriction sites is shown (Patent Document 1 reference, hereinafter, 'the conventional example 2' that). In addition, Fig. 1C is, in addition to the above conventional example 1, with a linker to a solid phase 1 for separating the first and second cleavage sites for the first 2 linker is shown (see Patent Document 1, hereinafter, 'the conventional example 3' referred to.). Is Fig. 1D, the main chain of 2 linker linked psoralen has been shown (see Patent Document 2, hereinafter, 'the conventional example 4' referred to.).
In addition to the above-described linker, is integrated into the main chain of the linker cnvK, light mRNA and cross-linking with the main chain of the linker that has been proposed (see Patent Document 3, hereinafter, 'the conventional example 5' referred to.).
However, the current Japanese leading cause of death in cancer (tumor), in the mixture is measured to its death, has been increasing year by. Whether or not suffering from a tumor, and the main treatment in order to observe the course after, and to exist in the tumor in the body, in blood or urine, or above a certain level of a particular substance (tumor marker) is detected as. Then, the type of tumor markers and their blood or urine by measuring the level, of whether or not the degree of progression of the tumor can be known.
Here, the tumor marker is, in most of the carbohydrate antigen. And normal cells by, the chain length is on the cell surface changes by glycosyltransferases, sugar chain specific for cancer cells, such sugar chain tumor markers for identifying cancer (hereinafter, 'sugar-chain tumor marker' is referred.) And (see non-patent document 5) are. Here, an antigenic determinant of a tumor marker sugar chain (hereinafter, referred to as' epitope '.) Is, schematically Fig. 2 (A) has a structure such as that depicted in general, from its structure, as shown in the following Table 1, 1-type sugar chain, 2-type sugar chain, sugar chain mother nucleus, is divided into core protein.
TABLE 1
Here, the 1 and 2 is-type sugar chain-type sugar chain, as shown in the above Table 1 the film is known, any of these, present on the cell surface sugar chain elongated by glycosyltransferase, are also longer than the case of normal cells. In addition, of the virus core protein and protein that is present around the nucleic acid.
(B) 2 to FIG., 1 of the backbone structure of a sugar chain structure of the type CA19-9(Carbohydrate Antigen 19-9) antigen, such as a carbohydrate antigen of example 1 schematically. Is CA19-9, 5 monosaccharide having the structure of 874 molecule of molecular weight, each monosaccharide is connected to the source by a glycosidic bond. In the figure, is NeuNac N- acetylneuraminic acid, gal is galactose, fucose Fuc, GlcNAc is N- acetyl glucosamine -D- respectively. Then, as shown in Fig. 2 (B) CA19-9 GlcNAc is included, other than the disk CA19-9 also included in the carbohydrate antigen is known (see non-patent document 5, hereinafter, 'the conventional example 6' referred to.).
In addition, the antibody, generally the light and heavy - chain and therefore, the molecular weight of the immunoglobulin (IgG), known as large as 170KDa. On the other hand, does not have a light chain of the camelidae antibody, and a heavy chain only, as small as 12KDa molecular weight, high plasticity and a three-dimensional structure, superior in thermal stability is known. Or less, such a region (domain) may also be referred to a molecule having a 'VHH'.
Scope of claims (In Japanese)[請求項1]
主鎖と側鎖とを有するリンカーであって、
前記主鎖は、
所定の塩基配列を有し、前記主鎖の5’末端に位置し固相との結合を形成するための固相結合部位と;
前記固相結合部位ごと前記固相を切り離すための固相切断部位と;
前記側鎖を連結するための側鎖連結部位と;
前記側鎖連結部位と前記固相切断部位との間に位置し、前記主鎖と相補的な配列を有するmRNAを、前記主鎖と光架橋によって連結する高速光架橋部位と;
前記側鎖連結部位に隣接し、前記主鎖の3’末端に位置する逆転写開始領域と;
を備え、
前記側鎖は、蛍光標識と、遊離末端に位置するタンパク質結合部位と、前記主鎖の側鎖連結部位に連結される連結形成部位とを備え、
前記側鎖は前記連結形成部位で前記側鎖連結部位に連結されている、試験管内淘汰及び分子間相互作用解析のための高速光架橋型共用リンカー。
[請求項2]
前記固相切断部位は、デオキシイノシン、リボG又はリボピリミジンからなる群から選ばれる塩基で構成される、ことを特徴とする請求項1に記載の試験管内淘汰及び分子間相互作用解析のための高速光架橋型共用リンカー。
[請求項3]
前記高速光架橋部位はシアノビニルカルバゾール化合物で構成される、ことを特徴とする請求項1又は2に記載の試験管内淘汰及び分子間相互作用解析のための高速光架橋型共用リンカー。
[請求項4]
前記シアノビニルカルバゾール化合物は3-シアノビニルカルバゾールである、ことを特徴とする請求項3に記載の試験管内淘汰及び分子間相互作用解析のための高速光架橋型共用リンカー。
[請求項5]
前記固相結合部位は、ビオチン、ストレプトアビジン、アルキン、クリックケミストリーによるアジ化物、アミノ基、N-ヒドロキシスクシンイミドエステル(NHS)、SH基、及びAuからなる群から選ばれるいずれかの化合物と、前記化合物に結合したポリAとで構成される、ことを特徴とする請求項1~4のいずれかに記載の試験管内淘汰及び分子間相互作用解析のための高速光架橋型共用リンカー。
[請求項6]
前記タンパク質結合部位はピューロマイシン又はその類縁化合物で構成されている、ことを特徴とする請求項1~5のいずれかに記載の試験管内淘汰及び分子間相互作用解析のための高速光架橋型共用リンカー。
[請求項7]
前記ピューロマイシンの類縁化合物は、3’-N-アミノアシルピューロマイシン及び3’-N-アミノアシルアデノシンアミノ酸のヌクレオシドからなる群から選ばれるいずれかの化合物である、ことを特徴とする請求項6に記載の試験管内淘汰及び分子間相互作用解析のための高速光架橋型共用リンカー。
[請求項8]
請求項1に記載の試験管内淘汰及び分子間相互作用解析のための高速光架橋型共用リンカーの主鎖と所望のmRNAとを相補結合させる相補結合形成工程と;
前記相補結合が形成された主鎖とmRNAとに、300~400nmの波長の光を0.5~5分間照射して光架橋を形成させる光架橋工程と;
前記リンカーに結合されたmRNAを無細胞翻訳系で翻訳し、前記mRNAに対応するタンパク質を前記リンカーのタンパク質結合部位に結合させ、mRNA-タンパク質の融合体を形成する融合体形成工程と;
前記融合体を固相に結合させる固相結合工程と;
前記融合体に含まれるmRNAを逆転写し、前記融合体と逆転写されたcDNAとの結合体を形成する逆転写工程と;
前記結合体を固相切断部位から切断し、所望のcDNAディスプレイ法を選択する選択工程と;
を備える、試験管内淘汰方法。
[請求項9]
前記固相は、ストレプトアビジン又はアビジンでコーティングされた磁性粒子である、ことを特徴とする請求項8に記載の試験管内淘汰方法。
[請求項10]
前記選択工程における前記結合体の切断は、エンドヌクレアーゼV、RNase T1、及びRNase Aからなる群から選ばれるいずれかの酵素で行う、ことを特徴とする請求項8又は9に記載の試験管内淘汰方法。
[請求項11]
前記高速光架橋型共用リンカーの主鎖は、糖鎖抗原を認識する配列を有する、ことを特徴とする、請求項8~10のいずれかに記載の試験管内淘汰方法。
[請求項12]
請求項1に記載の試験管内淘汰及び分子間相互作用解析のための高速光架橋型共用リンカーの主鎖と所望のmRNAとを相補結合させる相補結合形成工程と;
前記相補結合が形成された主鎖とmRNAとに、300~400nmの波長の光を0.5~5分間照射して光架橋を形成させる光架橋工程と;
前記リンカーに結合されたmRNAを無細胞翻訳系で翻訳し、前記mRNAに対応するタンパク質を前記リンカーのタンパク質結合部位に結合させ、mRNA-タンパク質の融合体を形成する融合体形成工程と;
前記融合体をRNAで消化してリンカー-タンパク質融合体とする、リンカー-タンパク質融合体形成工程と;
前記リンカー-タンパク質融合体を固相に結合させる固相結合工程と;
前記リンカー-タンパク質融合体を所定の条件下で前記固相から溶出させ、精製する精製工程と;
を備える親和性測定用リンカー-タンパク質の作製方法。
[請求項13]
前記固相は、ストレプトアビジン又はアビジンでコーティングされた磁性粒子である、ことを特徴とする請求項12に記載の親和性測定用リンカー-タンパク質の作製方法。
[請求項14]
前記精製工程は、0~100mM NaClを含む水溶液中、室温にて行う、ことを特徴とする請求項12又は13に記載の親和性測定用リンカー-タンパク質の作製方法。
[請求項15]
請求項12~14のいずれかに記載の方法で作製された親和性測定用リンカー-タンパク質。
  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • SAITAMA UNIVERSITY
  • Inventor
  • NEMOTO, Naoto
  • MOCHIZUKI, Yuki
  • KUMACHI, Shigefumi
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 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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