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- JPWO2020235635A5 JPWO2020235635A5 JP2021520845A JP2021520845A JPWO2020235635A5 JP WO2020235635 A5 JPWO2020235635 A5 JP WO2020235635A5 JP 2021520845 A JP2021520845 A JP 2021520845A JP 2021520845 A JP2021520845 A JP 2021520845A JP WO2020235635 A5 JPWO2020235635 A5 JP WO2020235635A5
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- 108091008104 nucleic acid aptamers Proteins 0.000 claims description 23
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 claims description 20
- 150000001768 cations Chemical class 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 12
- 210000000170 cell membrane Anatomy 0.000 claims description 9
- 210000004027 cell Anatomy 0.000 claims description 7
- 108091033319 polynucleotide Proteins 0.000 claims description 6
- 102000040430 polynucleotide Human genes 0.000 claims description 6
- 239000002157 polynucleotide Substances 0.000 claims description 6
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 4
- 230000009870 specific binding Effects 0.000 claims description 4
- 230000035699 permeability Effects 0.000 claims description 3
- 239000002773 nucleotide Substances 0.000 claims 2
- 125000003729 nucleotide group Chemical group 0.000 claims 2
- 230000005540 biological transmission Effects 0.000 claims 1
- 238000010586 diagram Methods 0.000 description 4
- 108091008102 DNA aptamers Proteins 0.000 description 3
- 238000001142 circular dichroism spectrum Methods 0.000 description 2
- 239000012466 permeate Substances 0.000 description 2
- 229910001414 potassium ion Inorganic materials 0.000 description 2
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 101000742054 Homo sapiens Protein phosphatase 1D Proteins 0.000 description 1
- NPYPAHLBTDXSSS-UHFFFAOYSA-N Potassium ion Chemical compound [K+] NPYPAHLBTDXSSS-UHFFFAOYSA-N 0.000 description 1
- 102100038675 Protein phosphatase 1D Human genes 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000027455 binding Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Description
【０００２】
［０００５］
特許文献１：米国特許第６０１１０２０号明細書
発明の概要
発明が解決しようとする課題
［０００６］
本発明は、上記事情に鑑みてなされたものであって、細胞内の任意の標的分子に対する結合能及び細胞膜透過能を時空間的に制御可能である核酸アプタマーを提供する。
課題を解決するための手段
［０００７］
本発明者らは、上記目的を達成すべく鋭意研究を重ねた結果、グアニンリッチなＤＮＡ分子が陽イオンに応答し四重鎖構造を形成することに着目し、細胞内の任意の標的分子に対して高い特異性及び選択性を有し、且つ陽イオン刺激により細胞内の任意の標的分子への結合能及び細胞膜透過能を時空間的に制御できる核酸アプタマーを見出し、本発明を完成するに至った。
［０００８］
すなわち、本発明は、以下の態様を含む。
本発明の第１態様に係る方法は、陽イオン存在下でグアニン四重鎖構造を形成し、細胞膜透過能を有する、核酸アプタマーを、陽イオン存在下で、導入剤を使用せずに細胞と接触させることを含む、前記核酸アプタマーの細胞膜透過方法であって、
前記核酸アプタマーが、
グアニン四重鎖構造を形成する第１領域、第２領域、第３領域及び第４領域と、
前記第１領域、前記第２領域、前記第３領域及び前記第４領域のそれぞれの間に連結領域Ａ、連結領域Ｂ及び連結領域Ｃと、を有し、
前記第１領域は配列番号１に示される塩基配列からなり、
前記第２領域、前記第３領域及び前記第４領域はそれぞれ配列番号２に示される塩基配列からなり、
前記連結領域Ａ、前記連結領域Ｂ及び前記連結領域Ｃからなる群より選ばれる少なくとも１つの領域は、陽イオン存在下で標的分子への特異的な結合能を有する。0002.
[0005]
Patent Document 1: US Pat. No. 6,011,020 Outline of the invention Problem to be solved by the invention [0006]
The present invention has been made in view of the above circumstances, and provides a nucleic acid aptamer capable of spatiotemporally controlling the ability to bind to an arbitrary target molecule in a cell and the ability to permeate a cell membrane.
Means for Solving Problems [0007]
As a result of diligent research to achieve the above objectives, the present inventors focused on the fact that guanine-rich DNA molecules respond to cations to form a quadruplex structure, and used them as arbitrary target molecules in cells. To complete the present invention, a nucleic acid aptamer having high specificity and selectivity and capable of spatiotemporally controlling the ability to bind to an arbitrary target molecule in a cell and the ability to permeate a cell membrane by cation stimulation has been found. I arrived.
[0008]
That is, the present invention includes the following aspects.
In the method according to the first aspect of the present invention, a nucleic acid aptamer that forms a guanine quadruplex structure in the presence of cations and has cell membrane permeability can be used with cells in the presence of cations without using an introducer. A method for permeating the cell membrane of the nucleic acid aptamer, which comprises contacting the nucleic acid aptamer.
The nucleic acid aptamer
The first region, the second region, the third region, and the fourth region forming the guanine quadruple chain structure,
A connecting region A, a connecting region B, and a connecting region C are provided between the first region, the second region, the third region, and the fourth region, respectively.
The first region consists of the base sequence shown in SEQ ID NO: 1.
The second region, the third region, and the fourth region each consist of the base sequence shown in SEQ ID NO: 2.
At least one region selected from the group consisting of the linking region A, the linking region B, and the linking region C has a specific binding ability to a target molecule in the presence of a cation.
【０００３】
前記陽イオンが特定の種類の陽イオンであってもよい。
上記第１態様に係る方法において、前記核酸アプタマーは、前記第４領域の下流に、３’末端付加配列を更に有し、前記３’末端付加配列は、配列番号３に示される塩基配列からなってもよい。
上記第１態様に係る方法において、前記核酸アプタマーは、配列番号４に示される塩基配列からなるポリヌクレオチドを含んでもよい。
上記第１態様に係る方法において、前記核酸アプタマーは、配列番号５～７のいずれかに示される塩基配列からなるポリヌクレオチドを含んでもよい。
本発明の第２態様に係る核酸アプタマーは、陽イオン存在下でグアニン四重鎖構造を形成し、細胞膜透過能を有する、核酸アプタマーであって、
グアニン四重鎖構造を形成する第１領域、第２領域、第３領域及び第４領域と、
前記第１領域、前記第２領域、前記第３領域及び前記第４領域のそれぞれの間に連結領域Ａ、連結領域Ｂ及び連結領域Ｃと、を有し、
前記第１領域は配列番号１に示される塩基配列からなり、
前記第２領域、前記第３領域及び前記第４領域はそれぞれ配列番号２に示される塩基配列からなり、
前記連結領域Ａ、前記連結領域Ｂ及び前記連結領域Ｃからなる群より選ばれる少なくとも１つの領域は、陽イオン存在下で標的分子への特異的な結合能を有し、
配列番号５～７のいずれかに示される塩基配列からなるポリヌクレオチドを含む。
発明の効果
［０００９］
上記態様の核酸アプタマーによれば、細胞内の任意の標的分子に対する結合能及び細胞膜透過能を時空間的に制御可能である核酸アプタマーを提供することができる。
図面の簡単な説明
［００１０］
［図１］グアニン四重鎖構造の分類型を示す図である。
［図２Ａ］本発明の一実施形態に係る核酸アプタマーを模式的に示す図である。
［図２Ｂ］本発明の他の実施形態に係る核酸アプタマーを模式的に示す図である。
［図３］実施例１における各ＤＮＡアプタマーの塩基配列を模式的に示す図である。
［図４］実施例１における各ＤＮＡアプタマーによるＰＰＭ１Ｄの阻害曲線を示すグラフである。
［図５］実施例１における異なる濃度のカリウムイオン存在下での各ＤＮＡアプタマーの円偏光二色性（Ｃｉｒｃｕｌａｒ Ｄｉｃｈｒｏｉｓｍ；ＣＤ）スペクトルを示すグラフである。
［図６］図５に示すＣＤスペクトルにおけるピーク波長（Ｍ１Ｄ－Ｑ５Ｆ：２６７ｎｍ、Ｍ１Ｄ－Ｑ５Ｍ及びＭ１Ｄ－Ｑ５：２６５ｎｍ）でのカリウムイオンの濃度の違いによる変化を解析したグラフである。
［図７］実施例１におけるＭ１Ｄ－Ｑ５Ｆ又はＭ１Ｄ－Ｑ５ＭをＡｕｔｏ－ｐａｎｅｔｒａｔｉｏｎ法で投与したＭＣＦ７細胞でのｐ５３及びβ－アクチンのタンパク質発現量をウエスタンブロッティング法で解析した結果を示す画像である。
［図８］図７に示すバンドのシグナルを定量化したグラフである。0003
The cation may be a specific type of cation.
In the method according to the first aspect, the nucleic acid aptamer further has a 3'end-end addition sequence downstream of the fourth region, and the 3'end-end addition sequence comprises the base sequence shown in SEQ ID NO: 3. You may.
In the method according to the first aspect, the nucleic acid aptamer may contain a polynucleotide consisting of the base sequence shown in SEQ ID NO: 4.
In the method according to the first aspect, the nucleic acid aptamer may contain a polynucleotide consisting of the base sequence shown in any of SEQ ID NOs: 5 to 7.
The nucleic acid aptamer according to the second aspect of the present invention is a nucleic acid aptamer that forms a guanine quadruplex structure in the presence of cations and has a cell membrane penetrating ability.
The first region, the second region, the third region, and the fourth region forming the guanine quadruple chain structure,
A connecting region A, a connecting region B, and a connecting region C are provided between the first region, the second region, the third region, and the fourth region, respectively.
The first region consists of the base sequence shown in SEQ ID NO: 1.
The second region, the third region, and the fourth region each consist of the base sequence shown in SEQ ID NO: 2.
At least one region selected from the group consisting of the linking region A, the linking region B, and the linking region C has a specific binding ability to a target molecule in the presence of a cation.
It contains a polynucleotide consisting of the base sequence shown in any of SEQ ID NOs: 5 to 7.
Effect of the invention [0009]
According to the nucleic acid aptamer of the above aspect, it is possible to provide a nucleic acid aptamer capable of spatiotemporally controlling the binding ability and the cell membrane permeation ability to any target molecule in the cell.
Brief Description of Drawings [0010]
FIG. 1 is a diagram showing a classification type of a guanine quadruple chain structure.
FIG. 2A is a diagram schematically showing a nucleic acid aptamer according to an embodiment of the present invention.
FIG. 2B is a diagram schematically showing a nucleic acid aptamer according to another embodiment of the present invention.
FIG. 3 is a diagram schematically showing the base sequence of each DNA aptamer in Example 1.
FIG. 4 is a graph showing the inhibition curve of PPM1D by each DNA aptamer in Example 1.
FIG. 5 is a graph showing Circular Dichroism (CD) spectra of each DNA aptamer in the presence of different concentrations of potassium ions in Example 1.
FIG. 6 is a graph analyzed for changes in potassium ion concentration at peak wavelengths (M1D-Q5F: 267 nm, M1D-Q5M and M1D-Q5: 265 nm) in the CD spectrum shown in FIG.
FIG. 7 is an image showing the results of analysis of p53 and β-actin protein expression levels in MCF7 cells administered with M1D-Q5F or M1D-Q5M by the Auto-panetration method in Example 1 by Western blotting.
FIG. 8 is a graph quantifying the signal of the band shown in FIG. 7.
Claims (6)
前記核酸アプタマーが、
グアニン四重鎖構造を形成する第１領域、第２領域、第３領域及び第４領域と、
前記第１領域、前記第２領域、前記第３領域及び前記第４領域のそれぞれの間に連結領域Ａ、連結領域Ｂ及び連結領域Ｃと、を有し、
前記第１領域は配列番号１に示される塩基配列からなり、
前記第２領域、前記第３領域及び前記第４領域はそれぞれ配列番号２に示される塩基配列からなり、
前記連結領域Ａ、前記連結領域Ｂ及び前記連結領域Ｃからなる群より選ばれる少なくとも１つの領域は、陽イオン存在下で標的分子への特異的な結合能を有する、方法。The cell membrane of the nucleic acid aptamer, which comprises contacting a nucleic acid aptamer, which forms a guanine quadruplex structure in the presence of a cation and has cell membrane permeability, with a cell in the presence of a cation without the use of a introducer. It is a transmission method,
The nucleic acid aptamer
The first region, the second region, the third region, and the fourth region forming the guanine quadruple chain structure,
A connecting region A, a connecting region B, and a connecting region C are provided between the first region, the second region, the third region, and the fourth region, respectively.
The first region consists of the base sequence shown in SEQ ID NO: 1.
The second region, the third region, and the fourth region each consist of the base sequence shown in SEQ ID NO: 2.
A method in which at least one region selected from the group consisting of the connection region A, the connection region B and the connection region C has a specific binding ability to a target molecule in the presence of a cation.
前記３’末端付加配列は、配列番号３に示される塩基配列からなる、請求項１又は２に記載の方法。The nucleic acid aptamer further has a 3'end addition sequence downstream of the fourth region.
The method according to claim 1 or 2, wherein the 3'end addition sequence comprises the base sequence shown in SEQ ID NO: 3.
グアニン四重鎖構造を形成する第１領域、第２領域、第３領域及び第４領域と、
前記第１領域、前記第２領域、前記第３領域及び前記第４領域のそれぞれの間に連結領域Ａ、連結領域Ｂ及び連結領域Ｃと、を有し、
前記第１領域は配列番号１に示される塩基配列からなり、
前記第２領域、前記第３領域及び前記第４領域はそれぞれ配列番号２に示される塩基配列からなり、
前記連結領域Ａ、前記連結領域Ｂ及び前記連結領域Ｃからなる群より選ばれる少なくとも１つの領域は、陽イオン存在下で標的分子への特異的な結合能を有し、
配列番号５～７のいずれかに示される塩基配列からなるポリヌクレオチドを含む、核酸アプタマー。A nucleic acid aptamer that forms a guanine quadruple chain structure in the presence of cations and has cell membrane permeability.
The first region, the second region, the third region, and the fourth region forming the guanine quadruple chain structure,
A connecting region A, a connecting region B, and a connecting region C are provided between the first region, the second region, the third region, and the fourth region, respectively.
The first region consists of the base sequence shown in SEQ ID NO: 1.
The second region, the third region, and the fourth region each consist of the base sequence shown in SEQ ID NO: 2.
At least one region selected from the group consisting of the linking region A, the linking region B, and the linking region C has a specific binding ability to a target molecule in the presence of a cation.
A nucleic acid aptamer comprising a polynucleotide consisting of the nucleotide sequence set forth in any of SEQ ID NOs: 5-7.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2019096035 | 2019-05-22 | ||
| JP2019096035 | 2019-05-22 | ||
| PCT/JP2020/020119 WO2020235635A1 (en) | 2019-05-22 | 2020-05-21 | Nucleic acid aptamer |
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| Publication Number | Publication Date |
|---|---|
| JPWO2020235635A1 JPWO2020235635A1 (en) | 2020-11-26 |
| JPWO2020235635A5 true JPWO2020235635A5 (en) | 2022-05-17 |
| JP7477886B2 JP7477886B2 (en) | 2024-05-02 |
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| JP (1) | JP7477886B2 (en) |
| WO (1) | WO2020235635A1 (en) |
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