JP2020100632A - Quadruplex DNA detection probe and method for detecting quadruplex structure using the same - Google Patents
Quadruplex DNA detection probe and method for detecting quadruplex structure using the same Download PDFInfo
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- JP2020100632A JP2020100632A JP2020021535A JP2020021535A JP2020100632A JP 2020100632 A JP2020100632 A JP 2020100632A JP 2020021535 A JP2020021535 A JP 2020021535A JP 2020021535 A JP2020021535 A JP 2020021535A JP 2020100632 A JP2020100632 A JP 2020100632A
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Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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Abstract
Description
本発明は、四重らせんＤＮＡ検出プローブ及びそれを用いた四重らせん構造の検出方法並びに四重らせん結合物質のスクリーニング方法並びに四重らせん構造を持つＤＮＡデコイ分子に関する。 TECHNICAL FIELD The present invention relates to a quadruplex DNA detection probe, a method for detecting a quadruplex structure using the probe, a method for screening a quadruplex binding substance, and a DNA decoy molecule having a quadruplex structure.
ゲノムＤＮＡ中には、ＤＮＡが四重らせん(G-quadruplex)構造を持っている箇所が種々存在することが知られている。これらの四重らせん構造は、重要な生物学的メカニズムに関与している。例えば、四重らせん構造の形成は、テロメアを大幅に短縮させ、続いて、テロメアの末端に結合しているＴＲＦ２及び／又はＰｏｔ１が解離することにより、がん細胞のアポトーシスを引き起こすものである。また、ｃ−ｋｉｔとｃ−ｍｙｃの転写活性が、プロモーター領域での四重らせん構造の形成により抑制されることが、インビトロの実験で示されている。従って、強力で特異的に四重らせん構造に結合する物質は、生物学的ツールとなるだけでなく、抗がん剤の候補となりうるため、四重らせん構造に強力に結合する物質の開発を目的とした研究が盛んに行われている。 It is known that there are various locations in the genomic DNA where the DNA has a quadruplex structure. These quadruplex structures are involved in important biological mechanisms. For example, the formation of a quadruplex structure causes a shortening of telomeres, followed by dissociation of TRF2 and/or Pot1 bound to the ends of telomeres, thereby causing apoptosis of cancer cells. Further, in vitro experiments have shown that the transcriptional activities of c-kit and c-myc are suppressed by the formation of a quadruplex structure in the promoter region. Therefore, a substance that strongly and specifically binds to a quadruplex structure is not only a biological tool but also a candidate for an anticancer drug. Research aimed at is being actively conducted.
本出願人は、先に、四重らせん構造に特異的に結合して四重らせん構造を検出することができる、四重らせん構造の検出プローブを開発し、特許出願した（特許文献１）。 The present applicant has previously developed and applied for a patent for a detection probe of a quadruplex structure that can specifically bind to the quadruplex structure and detect the quadruplex structure (Patent Document 1).
本発明の目的は、新規な四重らせん構造検出プローブを提供し、それを用いてゲノムワイドに四重らせん構造を形成する部位を同定することである。また、本発明の目的は、このようにして同定された四重らせん構造部位に結合する物質をスクリーニングする方法を提供することである。さらに本発明の目的は、このようにして同定された四重らせん構造部位と他の物質との結合を競合的に阻害する、ＤＮＡデコイ分子を提供することである。 An object of the present invention is to provide a novel probe for detecting a quadruplex structure, and to use it to identify a site forming a quadruplex structure in a genome-wide manner. Another object of the present invention is to provide a method for screening a substance that binds to the thus identified quadruplex structure site. A further object of the present invention is to provide a DNA decoy molecule that competitively inhibits the binding between the thus identified quadruplex structure site and other substances.
本願発明者らは、鋭意研究の結果、特許文献１記載の四重らせん構造検出プローブにおける蛍光色素を、特許文献１には記載も示唆もされていない蛍光色素に変更した四重らせん構造検出プローブを用いることにより、ゲノムワイドに四重らせん構造を形成する部位を同定することに初めて成功し、本発明を完成した。 As a result of diligent research, the inventors of the present application have changed the fluorescent dye in the quadruplex structure detection probe described in Patent Document 1 to a fluorescent dye not described or suggested in Patent Document 1 The present invention was completed for the first time by identifying the site that forms a quadruplex structure in a genome-wide manner by using.
すなわち、本発明は、一般式[I]で表される化学構造を有する四重らせんＤＮＡ検出プローブを提供する。 That is, the present invention provides a quadruplex DNA detection probe having the chemical structure represented by the general formula [I].
式中、Ｘは、-CH2-、-NHC(NH)-、-CH(NH2)-、-C(NH)-、-NHCO-、-CO-、-NHCONHC(NH)-、シクロヘキサジエニレン基、又は、3,4,5,6-テトラヒドロピリミジン-2,6-イル基を表す。
Ｒ１は、単結合、炭素数１〜１２のアルキレン基、炭素数１〜１２のアルケニレン基又は炭素数１〜１２のアルキニレン基を表す。
Ｒ２、Ｒ３、Ｒ４、Ｒ５、Ｒ６及びＲ７は、それぞれ独立に、水素原子、アミノ基若しくはグアニジノ基を有していてもよい炭素数１〜１２のアルキル基、アミノ基、グアニジノ基、アミノ基若しくはグアニジノ基を有していてもよい炭素数７〜１２のアラルキル基、アミノ基若しくはグアニジノ基を有していてもよいフェニル基、炭素数２〜６の複素環基又はハロゲン原子を表す。
Ｒ8は単結合、炭素数１〜１２のアルキレン基、炭素数１〜１２のアルケニレン基又は炭素数１〜１２のアルキニレン基を表す。
Ｑはシアニン蛍光色素を表す。
In the formula, X is -CH 2 -, -NHC(NH)-, -CH(NH 2 )-, -C(NH)-, -NHCO-, -CO-, -NHCONHC(NH)-, cyclohexadi It represents an enylene group or a 3,4,5,6-tetrahydropyrimidin-2,6-yl group.
R 1 represents a single bond, an alkylene group having 1 to 12 carbon atoms, an alkenylene group having 1 to 12 carbon atoms, or an alkynylene group having 1 to 12 carbon atoms.
R 2 , R 3 , R 4 , R 5 , R 6 and R 7 are each independently a hydrogen atom, an alkyl group having 1 to 12 carbon atoms which may have an amino group or a guanidino group, an amino group, A guanidino group, an aralkyl group having 7 to 12 carbon atoms which may have an amino group or a guanidino group, a phenyl group which may have an amino group or a guanidino group, a heterocyclic group having 2 to 6 carbon atoms, or a halogen. Represents an atom.
R 8 represents a single bond, an alkylene group having 1 to 12 carbon atoms, an alkenylene group having 1 to 12 carbon atoms, or an alkynylene group having 1 to 12 carbon atoms.
Q represents a cyanine fluorescent dye.
また、本発明は、上記本発明のプローブと、被検ＤＮＡ又はその断片とを接触させ、ゲノムＤＮＡ又はその断片に結合したプローブを検出することを含む、被検ＤＮＡ中の四重らせん構造の検出方法を提供する。さらに本発明は、上記本発明の方法により検出された、四重らせん部位に結合し、該部位の近傍に位置する遺伝子の発現を増大又は減少させる物質をスクリーニングすることを含む、四重らせん結合物質のスクリーニング方法を提供する。さらに本発明は、配列番号１〜２０１８の各配列中に形成される四重らせん部位に結合する物質をスクリーニングすることを含む四重らせん結合物質のスクリーニング方法を提供する。さらに、本発明は、配列番号１〜２０１８に示される塩基配列と９０％以上の配列同一性を有し、四重らせん構造を持つ、ＤＮＡデコイ分子を提供する。さらに本発明は、配列番号１〜２０１８に示される塩基配列又は該塩基配列と９０％以上の配列同一性を有する塩基配列を持つ各ポリヌクレオチドの全部又は一部から成るライブラリーを作製し、該ライブラリーと標的物質とを接触させ、標的物質と結合したポリヌクレオチドを同定し、同定されたポリヌクレオチドを合成することを含む、標的物質と結合するアプタマーの製造方法を提供する。さらに本発明は、上記本発明のスクリーニング方法によりスクリーニングされた四重らせん結合物質を製造することを含む、四重らせん結合物質の製造方法を提供する。 The present invention also includes detecting a probe bound to genomic DNA or a fragment thereof by contacting the probe of the present invention with a test DNA or a fragment thereof, and detecting a probe having a quadruplex structure in the test DNA. Provide a detection method. Furthermore, the present invention provides a quadruplex binding, which comprises screening for a substance that binds to a quadruplex site detected by the method of the present invention and increases or decreases the expression of a gene located near the site. A method for screening a substance is provided. The present invention further provides a method for screening a quadruplex-binding substance, which comprises screening a substance that binds to the quadruplex region formed in each of SEQ ID NOS: 1 to 2018. Furthermore, the present invention provides a DNA decoy molecule having a quadruplex structure having 90% or more sequence identity with the nucleotide sequences shown in SEQ ID NOs: 1 to 2018. Furthermore, the present invention produces a library consisting of all or part of each polynucleotide having a nucleotide sequence represented by SEQ ID NO: 1 to 2018 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence, Provided is a method for producing an aptamer that binds to a target substance, which comprises contacting a library with a target substance, identifying a polynucleotide bound to the target substance, and synthesizing the identified polynucleotide. The present invention further provides a method for producing a quadruplex binding substance, which comprises producing the quadruplex binding substance screened by the screening method of the present invention.
本発明により、ゲノムワイドに四重らせん構造を形成する部位を同定することが可能な新規な四重らせん構造検出プローブが提供され、これを用いてゲノムワイドに四重らせん構造を形成する部位が初めて同定された。本発明により同定された四重らせん構造部位と結合する物質は、該四重らせん構造部位の近傍に位置する遺伝子の発現制御に影響を与える可能性が高く、有効な抗がん剤や有用な分子遺伝学の研究ツール等の候補となり得る。また、本発明により同定された四重らせん構造部位と同じ構造を持つＤＮＡデコイ分子は、該四重らせん構造部位と結合する物質と競合的に結合するものであるから、該四重らせん構造部位への該物質の結合を阻害するものであり、有効な抗がん剤や有用な分子遺伝学の研究ツール等の候補となり得る。 INDUSTRIAL APPLICABILITY The present invention provides a novel probe for detecting a quadruplex structure capable of identifying a site forming a quadruplex structure in a genome-wide region, and using this probe, a site forming a quadruplex structure in a genome-wide region is provided. First identified. The substance that binds to the quadruplex structure site identified by the present invention has a high possibility of affecting the expression control of a gene located near the quadruplex structure site, and is an effective anticancer agent or useful It can be a candidate as a research tool for molecular genetics. Further, the DNA decoy molecule having the same structure as the quadruplex structure site identified by the present invention binds competitively with a substance that binds to the quadruplex structure site. It inhibits the binding of the substance to the drug and can be a candidate for an effective anti-cancer drug or a useful research tool for molecular genetics.
上記の通り、本発明の四重らせん構造検出プローブは、上記一般式[I]で表される構造を有する。一般式[I]中、Ｑはシアニン蛍光色素を表す。シアニン蛍光色素は、蛍光を発する部分として、ポリメチン鎖を持つことを特徴とする一群の蛍光色素である。シアニン蛍光色素中のポリメチン部分の炭素数は通常、３〜７程度であり、５であるものが好ましい。特に、ポリメチン部分の炭素数が５である下記一般式[II]で表される構造を有するものが好ましい。 As described above, the quadruplex structure detection probe of the present invention has a structure represented by the above general formula [I]. In general formula [I], Q represents a cyanine fluorescent dye. Cyanine fluorescent dyes are a group of fluorescent dyes characterized by having a polymethine chain as a fluorescent moiety. The polymethine moiety in the cyanine fluorescent dye generally has about 3 to 7 carbon atoms, preferably 5 carbon atoms. Particularly, those having a structure represented by the following general formula [II] in which the polymethine moiety has 5 carbon atoms are preferable.
式中、Ｒ9は水素原子又は炭素数１〜６のアルキル基を示し、各円弧は、Ｎをヘテロ原子として含む環状構造（縮合環である場合を包含する）を示す。 In the formula, R 9 represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, and each arc represents a cyclic structure containing N as a hetero atom (including a condensed ring).
上記一般式[II]で表されるシアニン蛍光色素の中でも、下記一般式[III]で表されるものが好ましい。 Among the cyanine fluorescent dyes represented by the above general formula [II], those represented by the following general formula [III] are preferable.
式中、Ｒ9'は炭素数１〜６のアルキル基を示し、Ｒ10、Ｒ11、Ｒ12及びＲ13は互いに独立して水素原子又は炭素数１〜６のアルキル基を示す。 In the formula, R 9′ represents an alkyl group having 1 to 6 carbon atoms, and R 10 , R 11 , R 12 and R 13 each independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms.
下記実施例において具体的に用いたプローブは、一般式[III]中のＲ9'、Ｒ10、Ｒ11、Ｒ12及びＲ13が全てメチル基であるものである。 The probe specifically used in the following examples is a probe in which R 9′ , R 10 , R 11 , R 12 and R 13 in the general formula [III] are all methyl groups.
本発明のプローブにおいて、シアニン蛍光色素以外の部分の構造は、特許文献１に記載されているプローブの構造と同様であり、各符号の定義は上記した通りである。一般式[I]中のＸは、−ＣＨ２−又は−ＮＨＣ（ＮＨ）−とすることが好ましく、特に、−ＣＨ２−とすることが好ましい。Ｒ１は、炭素数１〜１２のアルキレン基とすることが好ましく、より好ましくは、炭素数１〜７の直鎖状のアルキレン基とすることが好ましい。Ｒ２、Ｒ３、Ｒ４、Ｒ５、Ｒ６及びＲ７は、それぞれ独立に、水素原子、アミノ基若しくはグアニジノ基を有していてもよい炭素数１〜７のアルキル基であることが好ましく、これらの全てが水素原子であるものが合成が容易で四重らせん構造との結合性も高く好ましい。Ｒ8は炭素数１〜７のアルキレン基であるものが好ましい。 In the probe of the present invention, the structure of the portion other than the cyanine fluorescent dye is the same as the structure of the probe described in Patent Document 1, and the definition of each symbol is as described above. X in general formula [I] is preferably —CH 2 — or —NHC(NH)—, and particularly preferably —CH 2 —. R 1 is preferably an alkylene group having 1 to 12 carbon atoms, and more preferably a linear alkylene group having 1 to 7 carbon atoms. R< 2 >, R< 3 >, R< 4 >, R< 5 >, R< 6 > and R< 7 > are each independently a hydrogen atom, an amino group or an alkyl group having 1 to 7 carbon atoms which may have a guanidino group. It is preferable that all of these are hydrogen atoms because the synthesis is easy and the bondability with the quadruplex structure is high. R 8 is preferably an alkylene group having 1 to 7 carbon atoms.
下記実施例において具体的に用いたプローブは、上記一般式[I]において、Ｘが−ＣＨ２−、Ｒ１が炭素数３のアルキレン基、Ｒ２、Ｒ３、Ｒ４、Ｒ５、Ｒ６及びＲ７が全て水素原子、Ｒ8が炭素数５のアルキレン基であるものである。 The probe specifically used in the following examples is a compound represented by the above general formula [I] in which X is —CH 2 —, R 1 is an alkylene group having 3 carbon atoms, R 2 , R 3 , R 4 , R 5 , and R 5 . 6 and R 7 are all hydrogen atoms, and R 8 is an alkylene group having 5 carbon atoms.
本発明の検出プローブは、シアニン蛍光色素以外の部分は、特許文献１に記載された方法により製造することができ、一方、シアニン蛍光色素自体は、他の化合物との結合が容易なものが種々公知であり、Cy5等の市販品も利用可能であるので、当業者が容易に製造することができ、下記実施例にも具体的な製造方法が記載されている。 The detection probe of the present invention, except for the cyanine fluorescent dye, can be produced by the method described in Patent Document 1, while the cyanine fluorescent dye itself has various substances that are easily bound to other compounds. Since it is publicly known and commercially available products such as Cy5 are also available, it can be easily produced by those skilled in the art, and specific production methods are also described in the following examples.
本発明の四重らせん構造検出プローブは、ＤＮＡの四重らせん構造部位に特異的に結合するので、本発明のプローブと、被検ＤＮＡ又はその断片とを接触させ、ゲノムＤＮＡ又はその断片に結合したプローブを検出することにより、被検ＤＮＡ中の四重らせん構造を検出することができる。被検ＤＮＡとしては、ゲノムＤＮＡ又はその断片が好ましく、特に多数のゲノムＤＮＡの断片をチップ上に固相化してＤＮＡチップとしたものが好ましい。四重らせん構造はプロモーター領域やCpG（シトシン(C)(5'側）とグアニン(G)が１個のリン酸エステル(p)を介して結合している部分）部位を複数含む領域(CpG islandと呼ばれる）に多く含まれることが予測されていることから、ゲノムＤＮＡのうち、プロモーターやCpG islandを含む断片だけをチップ上に固定化したものを対象として検出操作を行うことが効率的で望ましい。 Since the quadruplex structure detection probe of the present invention specifically binds to a quadruplex structure site of DNA, the probe of the present invention is contacted with a test DNA or a fragment thereof to bind to a genomic DNA or a fragment thereof. The quadruplex structure in the test DNA can be detected by detecting the probe. The test DNA is preferably genomic DNA or a fragment thereof, and particularly preferably a DNA chip obtained by immobilizing a large number of genomic DNA fragments on a chip. The quadruplex structure includes a promoter region and a region (CpG) that contains multiple CpG (sites in which cytosine (C) (5' side) and guanine (G) are bound via one phosphate ester (p)) site. It is expected that it will be efficiently contained in the genomic DNA, and it will be efficient to carry out the detection operation for genomic DNA in which only the fragment containing the promoter and CpG island is immobilized on the chip. desirable.
検出操作自体は、本発明の検出プローブを用いる点を除けば、公知の方法と同様であり、検出プローブを緩衝液中に溶解した溶液を、被検ＤＮＡと接触させ、インキュベーション後、洗浄し、洗浄後に被検ＤＮＡと結合している蛍光色素の蛍光を検出することにより行うことができる。具体的な操作方法は下記実施例に詳述されている。 The detection operation itself is the same as the known method except that the detection probe of the present invention is used, and a solution in which the detection probe is dissolved in a buffer solution is brought into contact with a test DNA, followed by incubation and washing, It can be performed by detecting the fluorescence of the fluorescent dye bound to the test DNA after washing. Specific operating methods are detailed in the examples below.
上記方法により蛍光が検出された部位は、本発明の検出プローブが結合している部位である。これらのうち、四重らせん構造をとるためには、グアニン(G)の２連続配列が４箇所以上必要であるので、検出された部位のうち、GGを４箇所以上含むものが四重らせん構造部位であると判定できる。実際、下記実施例において、検証した全ての配列において、四重らせん構造が確認された。 The site where fluorescence is detected by the above method is the site to which the detection probe of the present invention is bound. Of these, two or more guanine (G) consecutive sequences are required at four or more sites to form a quadruplex structure. Therefore, among the detected sites, those containing four or more GG sites have a quadruplex structure. It can be determined to be a part. In fact, a quadruplex structure was confirmed in all the verified sequences in the examples below.
マウスのCpG island及びヒトのCpG islandを含むゲノムＤＮＡを対象として上記方法を行った結果（下記実施例参照）、マウスについて1998配列、ヒトについて2018配列が四重らせん構造部位を含むものと同定された。同定された、ヒトのCpG islandを含む塩基配列を下記表１及び配列番号１〜２０１８に示す。また、表１には、各四重らせん構造部位の近傍に位置する遺伝子名も記載されている。ここで、「近傍に位置する」とは、プローブ配列から上流5kbp及び下流5kbp中に転写開始点が含まれる遺伝子を意味する。 As a result of performing the above method on genomic DNA containing mouse CpG island and human CpG island (see Examples below), it was identified that the 1998 sequence for mouse and the 2018 sequence for human contained a quadruplex structure site. It was The identified nucleotide sequences containing human CpG islands are shown in Table 1 below and SEQ ID NOS: 1 to 2018. In addition, Table 1 also describes the gene names located near each quadruplex structure site. Here, "located in the vicinity" means a gene having a transcription start point in 5 kbp upstream and 5 kbp downstream from the probe sequence.
このような四重らせん構造部位に結合する物質は、表１に記載されている各近傍遺伝子の機能発揮や発現に影響を与える可能性があり、有効な抗がん剤や有用な分子遺伝学の研究ツール等の候補となり得るものである。従って、本発明は、本発明の方法により検出された、四重らせん部位に結合する物質をスクリーニングすることを含む、四重らせん結合物質のスクリーニング方法をも提供するものである。また、本発明により、配列番号１〜２０１８の各配列が同定されたので、本発明は、これらの各配列中に形成される四重らせん部位に結合する物質をスクリーニングすることを含む四重らせん結合物質のスクリーニング方法をも提供するものである。なお、四重らせん構造部位に結合する物質のスクリーニングは、例えば、次のようにして行うことができる。各配列をFAM等、FRETドナーとなる蛍光団により修飾し、相補鎖をTAMRA等、FRETアクセプターとなる蛍光団により修飾し、このDNAをアニーリングし、種々の物質を加え、FRETが観察されなくなるものが、四重らせんに結合する物質として同定できる。（参考文献：Nucleic Acids Research、2011、Vol. 39、No. 4 e21） Substances that bind to such a quadruplex structure site may affect the function expression and expression of each neighboring gene listed in Table 1, and are effective anticancer agents and useful molecular genetics. It can be a candidate for research tools. Therefore, the present invention also provides a method for screening a quadruplex-binding substance, which comprises screening for a substance that binds to a quadruplex site detected by the method of the present invention. Further, since each sequence of SEQ ID NOS: 1 to 2018 has been identified by the present invention, the present invention includes a quadruple helix comprising screening a substance that binds to a quadruplex helix site formed in each of these sequences. It also provides a method for screening a binding substance. The substance that binds to the quadruplex structure can be screened as follows, for example. Each sequence is modified with a fluorophore that serves as a FRET donor, such as FAM, and the complementary strand is modified with a fluorophore that serves as a FRET acceptor, such as TAMRA, and this DNA is annealed and various substances are added so that FRET is no longer observed. Can be identified as a substance that binds to the quadruplex. (Reference: Nucleic Acids Research, 2011, Vol. 39, No. 4 e21)
あるいは、任意の標的物質に結合するアプタマーを、配列番号１〜２０１８又はこれらと類似する配列を持つポリヌクレオチドからスクリーニングすることもできる。すなわち、配列番号１〜２０１８に示される塩基配列又は該塩基配列と９０％以上の配列同一性（配列同一性の定義は後述）を有する塩基配列を持つ各ポリヌクレオチドの全部又は一部から成るライブラリーを作製し、該ライブラリーと標的物質とを接触させ、標的物質と結合したポリヌクレオチドを同定することにより、上記スクリーニングを行うことができる。より具体的に説明すると、本願発明者らは、これまでにプロモーター中に含まれる四重らせん構造を含む領域と同一の塩基配列を有する一本鎖ポリヌクレオチドが、そのままでアプタマーとして機能し、該アプタマーは、そのプロモーターが制御する構造遺伝子の遺伝子産物又は該遺伝子産物の関連遺伝子産物と結合することを見出している。例えば、VEGFA、PDGFA、RB1がそのプロモーター中に含まれる四重らせんDNAに結合することを示している。つまり、本発明で同定した配列番号１〜２０１８配列はその近傍に存在する遺伝子がコードするタンパク質に結合する可能性があるため、配列番号１〜２０１８に示される塩基配列又は該塩基配列と９０％以上の配列同一性（配列同一性の定義は後述）を有する塩基配列を持つ各ポリヌクレオチドの全部又は一部から成るライブラリーを用いれば、配列番号１〜２０１８の近傍に位置する遺伝子がコードするタンパク質及びその関連タンパク質に対するアプタマーを容易に取得できる。スクリーニングされたアプタマーは、市販のDNA合成機等を用いて容易に化学合成することができる。また、アプタマーは、酵素反応を利用した周知の核酸増幅法により合成することも可能であり、特にアプタマーがRNAである場合、酵素反応を利用した周知のRNA増幅法により合成することもできる。 Alternatively, aptamers that bind to any target substance can be screened from polynucleotides having SEQ ID NOs: 1 to 2018 or sequences similar thereto. That is, a live sequence consisting of all or part of each of the nucleotide sequences shown in SEQ ID NOs: 1 to 2018 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence (the definition of sequence identity will be described later). The above screening can be performed by preparing a rally, contacting the library with a target substance, and identifying the polynucleotide bound to the target substance. More specifically, the inventors of the present invention, a single-stranded polynucleotide having the same base sequence as the region containing the quadruplex structure contained in the promoter so far functions as an aptamer as it is, Aptamers have been found to bind to the gene product of a structural gene whose promoter controls or the related gene product of the gene product. For example, it is shown that VEGFA, PDGFA and RB1 bind to quadruplex DNA contained in its promoter. That is, since the SEQ ID NOS: 1 to 2018 identified in the present invention may bind to a protein encoded by a gene existing in the vicinity thereof, the nucleotide sequences shown in SEQ ID NOS: 1 to 2018 or 90% with the nucleotide sequences. Using a library consisting of all or part of each polynucleotide having a nucleotide sequence having the above sequence identity (the definition of sequence identity will be described later), genes located near SEQ ID NOs: 1 to 2018 encode An aptamer for a protein and its related protein can be easily obtained. The screened aptamer can be easily chemically synthesized using a commercially available DNA synthesizer or the like. Further, the aptamer can also be synthesized by a well-known nucleic acid amplification method utilizing an enzymatic reaction, and particularly when the aptamer is RNA, it can also be synthesized by a well-known RNA amplification method utilizing an enzymatic reaction.
また、特定のＤＮＡ領域に結合する物質が、該ＤＮＡ領域に結合することを競合的に阻害する物質としてＤＮＡデコイが知られている。ＤＮＡデコイは、対象となるＤＮＡ領域と、最も好ましくは同一の塩基配列を持つＤＮＡ分子であり、ＤＮＡデコイが細胞中に共存することにより、本来、そのＤＮＡ領域に結合すべき物質のうちの一部は、デコイ分子と結合するため、そのＤＮＡ領域と結合する物質の量を低減させることができる。従って、本発明はまた、配列番号１〜２０１８の各配列と９０％以上、好ましくは９５％以上、さらに好ましくは９９％以上、最も好ましくは１００％の配列同一性を有し、四重らせん構造を持つＤＮＡデコイ分子をも提供する。なお、ここで、配列同一性は、２つの配列を、一致塩基数が最大になるように並べ（ギャップが存在する場合にはギャップを入れる）、一致する塩基数を前塩基数（２つの配列の塩基数が異なる場合には大きい方の塩基数）で除した値であり、周知のプログラムにより容易に算出できる。 In addition, DNA decoy is known as a substance that competitively inhibits a substance that binds to a specific DNA region from binding to the DNA region. The DNA decoy is a DNA molecule most preferably having the same nucleotide sequence as the target DNA region, and one of the substances that should originally bind to the DNA region due to the coexistence of the DNA decoy in the cell. Since the part binds to the decoy molecule, the amount of the substance binding to the DNA region can be reduced. Therefore, the present invention also has 90% or more, preferably 95% or more, more preferably 99% or more, and most preferably 100% sequence identity with each of the sequences of SEQ ID NOs: 1 to 2018, and has a quadruplex structure. It also provides a DNA decoy molecule having Here, regarding the sequence identity, two sequences are arranged so that the number of matching bases is maximized (a gap is inserted if a gap exists), and the number of matching bases is determined by the number of previous bases (two sequences). When the number of bases is different, the value is divided by the larger number of bases), which can be easily calculated by a known program.
以下、本発明を実施例に基づきより具体的に説明する。もっとも、本発明は下記実施例に限定されるものではない。 Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.
実施例１
１． 検出プローブの作製
下記式で示される構造を有する四重らせん構造検出プローブ（L1Cy5-7OTD）を作製した。
Example 1
1. Preparation of Detection Probe A quadruplex structure detection probe (L1Cy5-7OTD) having a structure represented by the following formula was prepared.
すなわち、７つのオキサゾールからなる大環状構造を有し、側鎖としてＣｙ５基を有するL1Cy5-7OTDを、次のスキームに従って合成した。 That is, L1Cy5-7OTD having a macrocyclic structure composed of seven oxazoles and having a Cy5 group as a side chain was synthesized according to the following scheme.
（１）反応ａ
トリオキサゾール１（1070mg)を、ＴＨＦと水の混合溶媒(混合比率3:1 = v/v 30mL)に溶かし、一水和水酸化リチウム（98mg)を加えることにより、カルボン酸を生成させた。反応は、0℃で45分間行った。反応溶液をイオン交換樹脂Dowex50WX4（商品名）により中和し、樹脂を濾液により除き、濾液を精製することなく次の反応に用いた。
(1) Reaction a
Trioxazole 1 (1070 mg) was dissolved in a mixed solvent of THF and water (mixing ratio 3:1 = v/v 30 mL), and lithium monohydrate (98 mg) was added to generate a carboxylic acid. The reaction was carried out at 0° C. for 45 minutes. The reaction solution was neutralized with ion exchange resin Dowex 50WX4 (trade name), the resin was removed by the filtrate, and the filtrate was used in the next reaction without purification.
（２）反応ｂ
トリオキサゾール２（1020mg)を、水素をエアレーションしたテトラヒドロフラン（ＴＨＦ）とメタノールの混合溶媒(混合比率1:1 = v/v 30mL)に溶解し、水酸化パラジウム／炭素の存在下（30mg)に、ベンジルオキシカルボニル基（Ｃｂｚ）を脱離した。反応は、25℃で14時間行った。反応溶液を珪藻土により濾過し、濾液を精製することなく次の反応に用いた。
(2) Reaction b
Trioxazole 2 (1020 mg) was dissolved in a mixed solvent of tetrahydrofuran (THF) and methanol in which hydrogen was aerated (mixing ratio 1:1 = v/v 30 mL), and in the presence of palladium hydroxide/carbon (30 mg), The benzyloxycarbonyl group (Cbz) was eliminated. The reaction was carried out at 25°C for 14 hours. The reaction solution was filtered through diatomaceous earth, and the filtrate was used in the next reaction without purification.
（３）反応ｃ
トリオキサゾール１から合成したカルボン酸と、トリオキサゾール２から合成したアミンとを、ＴＨＦ、水およびＭｅＯＨの混合溶媒に溶かし、Ｎ−メチルモルホリン４−（４，６−ジメトキシ−１，３，５−トリアジン−２−イル）−４−メチルモルホルニウムクロライド（ＤＭＴ−ＭＭ）（986mg)とＮ−メチルモルホリン（ＮＭＭ）（400μL)を加えることにより、９１％の収率で直鎖状ヘキサオキサゾール３を1.64g得た。反応は、25℃で25時間行った。
(3) Reaction c
A carboxylic acid synthesized from trioxazole 1 and an amine synthesized from trioxazole 2 were dissolved in a mixed solvent of THF, water and MeOH, and N-methylmorpholine 4-(4,6-dimethoxy-1,3,5- Triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (986 mg) and N-methylmorpholine (NMM) (400 μL) were added to give linear hexaxazole 3 in 91% yield. Was obtained 1.64 g. The reaction was carried out at 25°C for 25 hours.
（４）反応ｄ−ｆ
反応ａと同じ条件で、ヘキサオキサゾール３のＣｂｚ基を脱離させ、反応ｂと同じ条件で、ヘキサオキサゾール３のメチルエステルを脱保護した。その後、生じたアミノ酸を、ジイソプロピルエチルアミン（ＥｔｉＰｒ２Ｎ） (250μL)、４−ジメチルアミノピリジン（ＤＭＡＰ）（179mg)及びジフェニルリン酸アジド（ＤＰＰＡ）（320μL)を加えたＮ‘Ｎ−ジメチルホルムアミドとジクロロメタン(1:2 = v/v 100mL)の混合溶媒中に、３ｍＭとなるよう高希釈して環化し、ヘキサオキサゾール３から６９％の収率で大環状ビスアミド４を179mg得た。反応は、25℃で3日間行った。
(4) Reaction df
The Cbz group of hexaoxazole 3 was eliminated under the same conditions as in Reaction a, and the methyl ester of hexaoxazole 3 was deprotected under the same conditions as in Reaction b. Then, the resulting amino acid was added to diisopropylethylamine (Et i Pr 2 N) (250 μL), 4-dimethylaminopyridine (DMAP) (179 mg) and diphenylphosphoric acid azide (DPPA) (320 μL) to prepare N′N-dimethyl. Cyclization was performed by highly diluting it in a mixed solvent of formamide and dichloromethane (1:2 = v/v 100 mL) to 3 mM to obtain 179 mg of macrocyclic bisamide 4 from hexaoxazole 3 in a yield of 69%. The reaction was carried out at 25°C for 3 days.
（５）反応ｇ−ｉ
大環状ビスアミド４（228mg)をＴＨＦ (40mL)に溶かし、ＨＦ・ピリジン (1.6mL)により、ｔｅｒｔ−ブチルジメチルシリル（ＴＢＳ）基を脱保護し、メタンスルホニルクロリド（ＭｓＣｌ）（110μL)とトリエチルアミン（Ｅｔ３Ｎ）（300μL)によるメシレーション後（25℃、1時間）、ジアザビシクロウンデセン（ＤＢＵ）（420μL)の処理（25℃、1時間）により９６％の収率でオレフィン５を186mg得た。
(5) Reaction g-i
Macrocyclic bisamide 4 (228 mg) was dissolved in THF (40 mL), tert-butyldimethylsilyl (TBS) group was deprotected with HF·pyridine (1.6 mL), and methanesulfonyl chloride (MsCl) (110 μL) and triethylamine ( After mesylation with Et 3 N (300 μL) (25° C., 1 hour), treatment with diazabicycloundecene (DBU) (420 μL) (25° C., 1 hour) provided 186 mg of olefin 5 in 96% yield. Obtained.
（７）反応ｊ
アセトニトリル溶媒中において、オレフィン５（25mg)をＮ−ブロムスクシンイミド（ＮＢＳ）（7mg)、さらに炭酸セシウム（59mg)により環化して（65℃、14時間）、３６％の収率でヘプタオキサゾール６を9mg得た。
(7) Reaction j
Olefin 5 (25 mg) was cyclized with N-bromosuccinimide (NBS) (7 mg) and cesium carbonate (59 mg) in an acetonitrile solvent (65°C, 14 hours) to give heptoxazole 6 in a yield of 36%. 9 mg was obtained.
（８）反応ｋ及びｌ
クロロホルム (9.5mL) 溶媒中において、ヘプタオキサゾール６のｔｅｒｔ−ブトキシカルボニル（Ｂｏｃ）基をトリフルオロ酢酸（ＴＦＡ）（0.5mL)により脱保護し（25℃、24時間）、９９％の収率でアミンを得た。その後得られたアミン (19mg) をDMF (1mL) 中、炭酸水素ナトリウム (23mg) 中、蛍光色素であるＣｙ５と連結したスクシンイミド (25mg) と反応させた。反応は、６０℃で３６時間行い、７８％の収率で生成物（Ｌ１Ｃｙ５−７ＯＴＤ）を21mg得た。
(8) Reactions k and l
The tert-butoxycarbonyl (Boc) group of heptoxazole 6 was deprotected with trifluoroacetic acid (TFA) (0.5 mL) in a solvent of chloroform (9.5 mL) (25°C, 24 hours), and the yield was 99%. The amine was obtained. The resulting amine (19 mg) was then reacted with succinimide (25 mg) linked to the fluorescent dye Cy5 in sodium hydrogen carbonate (23 mg) in DMF (1 mL). The reaction was carried out at 60°C for 36 hours to obtain 21 mg of the product (L1Cy5-7OTD) in a yield of 78%.
（９）ＮＭＲスペクトル測定
上記スキームにより合成されたＬ１Ｃｙ５−７ＯＴＤについて、重水素置換溶媒中において核磁気共鳴装置で測定を行い、ＮＭＲスペクトルを測定した。その結果は以下のとおりである。
(9) NMR spectrum measurement The L1Cy5-7OTD synthesized by the above scheme was measured by a nuclear magnetic resonance apparatus in a deuterium-substituted solvent to measure an NMR spectrum. The results are as follows.
1H NMR (500 MHz、ref 2.5 ppm for DMSO d-6)
δ9.05 (1H、s)、9.02 (1H、s)、8.99 (1H、s)、8.98 (1H、s)、8.97 (1H、s)、8.96 (1H、s)、8.84 (1H、s)、8.65-8.63 (1H、d、J = 7.45 Hz)、8.29-8.24 (2H、dd、J = 13.75、12.60 Hz)、7.72 (1H、m)、7.60-7.54 (2H、d、J = 7.45 Hz)、7.38-7.18 (4H、m)、6.48 (1H、d、J = 12.60 Hz)、6.24-6.21 (1H、d、J = 13.75 Hz)、6.20-6.17 (1H、d、J = 13.75 Hz)、5.51 (1H、m)、4.07 (1H、t、J = 7.45)、2.93 (2H、m)、2.02-1.05 (29H、m)
1 H NMR (500 MHz, ref 2.5 ppm for DMSO d-6)
δ 9.05 (1H, s), 9.02 (1H, s), 8.99 (1H, s), 8.98 (1H, s), 8.97 (1H, s), 8.96 (1H, s), 8.84 (1H, s) , 8.65-8.63 (1H, d, J = 7.45 Hz), 8.29-8.24 (2H, dd, J = 13.75, 12.60 Hz), 7.72 (1H, m), 7.60-7.54 (2H, d, J = 7.45 Hz) ), 7.38-7.18 (4H, m), 6.48 (1H, d, J = 12.60 Hz), 6.24-6.21 (1H, d, J = 13.75 Hz), 6.20-6.17 (1H, d, J = 13.75 Hz) , 5.51 (1H, m), 4.07 (1H, t, J = 7.45), 2.93 (2H, m), 2.02-1.05 (29H, m)
13C NMR (125 MHz、ref 49.0 ppm for DMSO d-6)
δ173.07、172.38、171.48、163.97、159.41、155.80、155.63、155.35、155.23、155.08、154.63、153.66、143.07、142.61、141.84、140.92、140.84、140.63、140.40、139.69、138.94、138.87、138.60、136.51、129.97、129.62、128.68、128.12、124.53、124.43、122.16、122.05、110.86、103.06、102.92、69.68、48.69、47.53、37.91、34.91、33.82、31.00、28.45、27.01、26.86、26.47、25.57、24.72、21.25.
13 C NMR (125 MHz, ref 49.0 ppm for DMSO d-6)
δ 173.07, 172.38, 171.48, 163.97, 159.41, 155.80, 155.63, 155.35, 155.23, 155.08, 154.63, 153.66, 143.07, 142.61, 141.84, 140.92, 140.84, 140.63, 140.40, 139.69, 138.94, 138.87, 138.60, 136.51, 136.51. 129.97, 129.62, 128.68, 128.12, 124.53, 124.43, 122.16, 122.05, 110.86, 103.06, 102.92, 69.68, 48.69, 47.53, 37.91, 34.91, 33.82, 31.00, 28.45, 27.01, 26.86, 26.47, 25.57.24.72, 24.72.
（１０）質量分析
上記スキームにより合成されたＬ１Ｃｙ５−７ＯＴＤについて、ＥＳＩ−ＭＳ装置にて測定を行い、分子量を測定したところ、分子量は1062.4304であった。また、得られた分子量値から元素組成を推定したところC59H56N11O9（理論値1062.4263）であった。この結果から目的の化合物が合成できたことを確認した。
(10) Mass Spectrometry The L1Cy5-7OTD synthesized by the above scheme was measured with an ESI-MS device to measure the molecular weight. As a result, the molecular weight was 1062.4304. Further, the elemental composition was estimated from the obtained molecular weight value and found to be C 59 H 56 N 11 O 9 (theoretical value 1062.4263). From this result, it was confirmed that the target compound could be synthesized.
（１１）ＨＰＬＣ測定
上記スキームにより合成されたＬ１Ｃｙ５−７ＯＴＤについて、逆相カラムを用いた分析を行い、純度を測定したところ９９％以上であった。
(11) HPLC measurement The L1Cy5-7OTD synthesized according to the above scheme was analyzed using a reverse phase column, and the purity was measured and found to be 99% or more.
実施例２ マウスゲノムＤＮＡ解析及び評価
１． マウスゲノムＤＮＡ解析
実施例１で作製した検出プローブを用いてマウスゲノムＤＮＡを解析した。被検ＤＮＡは、マウスゲノムＤＮＡ中のCpG islandのみ（約16000個）をチップ上に固相化したＤＮＡマイクロアレイ(Agilent社製G4811A)であった。具体的には以下の操作を行った。
Example 2 Mouse genomic DNA analysis and evaluation Mouse genomic DNA analysis Mouse genomic DNA was analyzed using the detection probe prepared in Example 1. The test DNA was a DNA microarray (G4811A manufactured by Agilent) in which only CpG islands (about 16000) in mouse genomic DNA were immobilized on a chip. Specifically, the following operations were performed.
(1) 終濃度10 nM L1Cy5-7OTDをBuffer A (50mM Tris-HCl (pH7.5)、100mM KCl、1×Hi-RPM Hybridization Buffer (Agilent))を用いて調製した。
(2) ガスケットスライド（Agilent）に(1)で調製した10 nM L1Cy5-7OTDを240μL加え、そこにMouse CpG island array (Agilent、G4811A)を乗せ、ハイブリチャンバーにセットとした。
(3) ハイブリチャンバーにセットしたアレイをハイブリゼーションオーブン(Agilent)にセットし、25℃、1時間、20 rpmでインキュベートした。
(3) 1時間後、ハイブリチャンバーを解体し、アレイをBuffer B (50mM Tris-HCl (pH7.5)、100mM KCl)中で5分間、25℃で洗浄した。
(4) 洗浄後、アレイをAgilent DNA Microarray scanner with surescan High resolution thchnology (Agilent)を用いてスキャンし、蛍光画像を得た。
(5) 得られた蛍光画像から、各プローブの蛍光強度(rProcessedSignal)及び塩基番号をAgilent Feature Extraction及びAgilent Genomic Workbenchを用いて抽出した。
(6) すべてのプローブの蛍光強度の平均値(394)に対して十分高い蛍光強度を示したプローブ(1705以上)をMicrosoft Excelにより抽出した。その結果、3122配列が抽出された。
(7) 抽出された3122配列の内、グアニンの2連続配列を4箇所以上含む配列のみ抽出した。その結果、1998配列を抽出することができた。
(1) A final concentration of 10 nM L1Cy5-7OTD was prepared using Buffer A (50 mM Tris-HCl (pH7.5), 100 mM KCl, 1×Hi-RPM Hybridization Buffer (Agilent)).
(2) To the gasket slide (Agilent), 240 μL of 10 nM L1Cy5-7OTD prepared in (1) was added, and the Mouse CpG island array (Agilent, G4811A) was placed on the gasket slide and set as a hybrid chamber.
(3) The array set in the hybrid chamber was set in a hybridization oven (Agilent) and incubated at 25°C for 1 hour at 20 rpm.
(3) After 1 hour, the hybrid chamber was disassembled, and the array was washed in Buffer B (50 mM Tris-HCl (pH 7.5), 100 mM KCl) for 5 minutes at 25°C.
(4) After washing, the array was scanned using Agilent DNA Microarray scanner with surescan High resolution thchnology (Agilent) to obtain a fluorescence image.
(5) The fluorescence intensity (rProcessedSignal) and base number of each probe were extracted from the obtained fluorescence image using Agilent Feature Extraction and Agilent Genomic Workbench.
(6) A probe (1705 or more) showing a sufficiently high fluorescence intensity with respect to the average value (394) of fluorescence intensity of all probes was extracted by Microsoft Excel. As a result, the 3122 sequence was extracted.
(7) Of the extracted 3122 sequences, only the sequence containing 4 or more 2 consecutive guanine sequences was extracted. As a result, the 1998 sequence could be extracted.
２．ルシフェラーゼレポーターアッセイ
上記１により同定された、四重らせん構造部位を持つ塩基配列のうち、いくつかのものについて、近傍遺伝子に対する影響をルシフェラーゼレポーターアッセイにより調べた。具体的には以下の操作を行った。
2. Luciferase Reporter Assay For some of the nucleotide sequences having a quadruplex structure site identified in 1 above, the effects on neighboring genes were examined by the luciferase reporter assay. Specifically, the following operations were performed.
(1) C57BL/6マウスゲノムDNAを鋳型として、Txndc5、Shd、Metrnl、Nup107、Rab1b、Smarcd1 CpG island (CGI)を以下の条件でPCR増幅した。 (1) Using C57BL/6 mouse genomic DNA as a template, Txndc5, Shd, Metrnl, Nup107, Rab1b, and Smarcd1 CpG island (CGI) were PCR-amplified under the following conditions.
(2) PCR産物をWizard（登録商標） SV Gel and PCR Clean-Up System(Promega)を用いて精製し、以下の組成でPCR産物とSfiI (NEB)を混合し、50℃2時間インキュベートすることによりPCR産物を切断した。 (2) Purify the PCR product using Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega), mix the PCR product and SfiI (NEB) with the following composition, and incubate at 50°C for 2 hours The PCR product was cleaved by.
(3) (2)と同様にホタルルシフェラーゼ遺伝子を持つpGL4.10[luc] vector (promega)をSfiIで切断した。組成は以下に示す。 (3) Similar to (2), pGL4.10[luc] vector (promega) containing the firefly luciferase gene was cut with SfiI. The composition is shown below.
(4) (2)、(3)で切断したPCR産物及びpGL4 vectorを1%アガロースを用いて電気泳動し、目的のバンドを切り出し、Wizard（商品名） SV Gel and PCR Clean-Up System(Promega)を用いて精製した。 (4) The PCR product cleaved in (2) and (3) and the pGL4 vector were electrophoresed using 1% agarose, the desired band was cut out, and the Wizard (trade name) SV Gel and PCR Clean-Up System (Promega ) Was used for purification.
(5) (4)で調製したpGL4 vector (18 fmol)、PCR産物(54 fmol)を混合し、Quickligation(NEB)を用いて5min 室温でライゲーションした。組成は以下に示す。 (5) The pGL4 vector (18 fmol) prepared in (4) and the PCR product (54 fmol) were mixed and ligated using Quickligation (NEB) at room temperature for 5 min. The composition is shown below.
(6) (5)で得られたライゲーションサンプルを7μL用い、70μLのJM110コンピテントセル(Agilent)に加え、30分氷上でインキュベートした。30分後、ヒートショック(42℃45秒インキュベート後、氷上で2分間インキュベート)を行い、得られた菌体をアンピシリンを含むLBプレートにプレーティングし、37℃で一晩培養した。
(7) 得られたコロニーをピックアップし、3mLのLB培地で37℃で一晩培養した。
(8) 得られた菌体からPureYield（商品名）Plasmid Miniprep System(promega)を用いてプラスミドを調製した。
(9) 得られたプラスミドをシークエンスし、ルシフェラーゼ遺伝子上流の目的の位置に標的CpG islandがクローニングされていることを確認した。
(10) 600 fmolの各プラスミドとコントロールベクターである12 fmolのウミシイタケルシフェラーゼ遺伝子発現vector pGL4.74(promega)を混合し、Opti-MEM(Invitrogen)を用いて300μLに調整し、6μL Lipofectoamine2000 (Invitrogen)と294μLのOpti-MEMを加え、20分間室温でインキュベートした。
(11)前日に2.5×104 cell/wellで24wellプレートに播種したNIH3T3細胞の培地を400μLのOpti-MEMに交換し、そこに(10)で調製したプラスミド混合溶液を100μLずつ6wellに加え、37℃、5% CO2条件下で4時間インキュベートした。
(12) 4時間後、1uM 7OTDを含む培地又は7OTDを含まない培地に交換し、20時間37℃、5% CO2条件下で培養した。
(13) 20時間後、Dual-Luciferase reporter Assay system (promega)を用いて、ホタルルシフェラーゼ及びウミシイタケルシフェラーゼの発光量を定量した。発光量はARVOMX 1420 Multilabel counter(Perkin Elmer)を用いて定量した。
(6) 7 μL of the ligation sample obtained in (5) was added to 70 μL of JM110 competent cells (Agilent), and incubated on ice for 30 minutes. After 30 minutes, heat shock (incubation at 42° C. for 45 seconds and then on ice for 2 minutes) was performed, and the obtained bacterial cells were plated on an LB plate containing ampicillin and cultured overnight at 37° C.
(7) The obtained colonies were picked up and cultured in 3 mL of LB medium at 37°C overnight.
(8) A plasmid was prepared from the obtained cells using PureYield (trade name) Plasmid Miniprep System (promega).
(9) The obtained plasmid was sequenced, and it was confirmed that the target CpG island was cloned at the target position upstream of the luciferase gene.
(10) 600 fmol of each plasmid and 12 fmol Renilla luciferase gene expression vector pGL4.74 (promega), which is a control vector, were mixed, adjusted to 300 μL using Opti-MEM (Invitrogen), and 6 μL Lipofectoamine 2000 (Invitrogen). ) And 294 μL of Opti-MEM were added and incubated for 20 minutes at room temperature.
(11) the medium of NIH3T3 cells seeded in a 24-well plate at 2.5 × 10 4 cells/well the day before was exchanged for 400 μL Opti-MEM, and the plasmid mixed solution prepared in (10) was added to 6 wells of 100 μL each, Incubated at 37°C, 5% CO 2 for 4 hours.
(12) After 4 hours, the medium was replaced with a medium containing 1 uM 7OTD or a medium not containing 7OTD, and cultured for 20 hours at 37° C. under 5% CO 2 condition.
(13) After 20 hours, the amount of luminescence of firefly luciferase and Renilla luciferase was quantified using the Dual-Luciferase reporter Assay system (promega). Luminescence was quantified using an ARVO MX 1420 Multilabel counter (Perkin Elmer).
(14) それぞれのホタルルシフェラーゼの発現量はコントロールベクターから発現したウミシイタケルシフェラーゼの発現量で標準化した。その結果、プロモーターをクローニングしていないベクターをトランスフェクションした場合と比較すると、Txndc5、Shd、Metrnl、Nup107、Rab1b、Smarcd1 CGIをクローニングした場合のホタルルシフェラーゼの発現量はそれぞれ7.9、2.9、0.5、0.8、517、34倍になった(表８)。つまり、Txndc5、Shd、Rab1b、Smarcd1 CGIはNIH 3T3細胞中でプロモーター活性を持つことが示された。1μM Gqリガンドを培地に加えるとTxndc5又はRab1b CGIをクローニングしたvectorではホタルルシフェラーゼの発現量の上昇が観察された。つまり、Txndc5 CGIとRab1b CGIはG4構造を形成することにより、転写活性が上昇することが示唆された。 (14) The expression level of each firefly luciferase was standardized by the expression level of Renilla luciferase expressed from the control vector. As a result, the expression levels of firefly luciferase when cloning Txndc5, Shd, Metrnl, Nup107, Rab1b, and Smarcd1 CGI were 7.9, 2.9, 0.5, and 0.8, respectively, as compared with the case of transfecting a vector in which the promoter was not cloned. , 517, and 34 times (Table 8). That is, Txndc5, Shd, Rab1b and Smarcd1 CGI were shown to have promoter activity in NIH 3T3 cells. When 1 μM Gq ligand was added to the medium, an increase in the expression level of firefly luciferase was observed in the vector in which Txndc5 or Rab1b CGI was cloned. In other words, it was suggested that Txndc5 CGI and Rab1b CGI form a G4 structure to increase transcriptional activity.
３． ゲルシフトアッセイ
１で同定された1998配列の中から配列（下記表９）をランダムに選択し、これら97配列が実際にL1Cy5-7OTDと結合するかゲルシフトアッセイを実施した。
3. Gel shift assay A sequence (Table 9 below) was randomly selected from the 1998 sequences identified in 1 and a gel shift assay was performed to determine whether these 97 sequences actually bind to L1Cy5-7OTD.
その結果、すべてのプローブDNAがL1Cy5-7OTDと結合していることが確認された。また、これら97配列がG4構造を形成しているかCDスペクトルを測定したところ、G4構造特有のCDスペクトルを示したことから、これらがG4構造を形成していることが確認された。なお、この操作は具体的には次のように行った。 As a result, it was confirmed that all probe DNAs were bound to L1Cy5-7OTD. In addition, when the CD spectrum was measured whether these 97 sequences form a G4 structure or not, a CD spectrum peculiar to the G4 structure was shown. Therefore, it was confirmed that they formed the G4 structure. In addition, this operation was specifically performed as follows.
(1) 500μM L1Cy5-7OTDを25 mM Tris-HCl (pH7.0)、250mM KClを用いて調製した。
(2) (1)で調製した500μM L1Cy5-7OTD 1.0μLに、25 mM Tris-HCl (pH7.0)、250mM KClを5.0μL、150mg/mL Ficol 400水溶液を2.0μL、種々の50μM DNAを2.0μL加え、これを電気泳動サンプルとした(L1Cy5-7OTDの終濃度は50μM、DNAの終濃度は10μM)。また、10 bp DNA ladder (Invitrogen) を、同量の150mg/mL Ficol 400水溶液と混合し、これを泳動用DNAラダーとした。
(3) 12%非変性ポリアクリルアミドゲルに、(2)で調製した電気泳動サンプル0.5μLおよび泳動用DNAラダー2.0μLをロードし、1×TBE bufferを用いて100 Vで10分間、その後200 Vで30分間電気泳動を行った。
(4) 泳動開始から40分後、ゲルをゲル板から外し、染色液A (1.0mg/mL Stains-all（商品名） ホルムアミド溶液200μLを染色バッファー（10% ホルムアミド、25% 2-プロパノール、65% 15 mM Tris (pH 8.8) 混合溶液100mL）に加えたもの) を用いて振とう撹拌により15分間染色した。
(5) 15分後、ゲルを染色液Aから取り出し、染色液B (10mg/mL エチジウムブロミド水溶液 10μLを1×TAE buffer 200mLに加えたもの) を用いて振とう撹拌により5分間染色した。
(6) 5分後、ゲルを染色液Bから取り出し、1×TBE bufferを用いて振とう撹拌により15分間脱色した。
(7) 脱色後、ゲルをTyphoon 8600 (GE Healthcare) を用いてスキャンし、ゲルの画像を得た。なお、フィルターは580-640 band pass filter（Stains-allおよびエチジウムブロミド検出用）および640-700 band pass filter（Cy5検出用）を用いた。
(8) DNAを染色した蛍光画像（緑）及びL1Cy5-7OTDの蛍光画像（赤）をImageJを用いてmergeし、L1Cy5-7OTDがDNAに結合しているか解析した。
(9)その結果、解析した97配列すべてがL1Cy5-7OTDと結合していることが確認できた。
(1) 500 μM L1Cy5-7OTD was prepared using 25 mM Tris-HCl (pH 7.0) and 250 mM KCl.
(2) To 500 μM L1Cy5-7OTD 1.0 μL prepared in (1), 25 mM Tris-HCl (pH 7.0), 250 mM KCl 5.0 μL, 150 mg/mL Ficol 400 aqueous solution 2.0 μL, various 50 μM DNA 2.0 μL was added, and this was used as an electrophoresis sample (final concentration of L1Cy5-7OTD was 50 μM, final concentration of DNA was 10 μM). Further, 10 bp DNA ladder (Invitrogen) was mixed with the same amount of 150 mg/mL Ficol 400 aqueous solution, and this was used as a DNA ladder for electrophoresis.
(3) Load 0.5 μL of the electrophoresis sample prepared in (2) and 2.0 μL of the DNA ladder for electrophoresis prepared in (2) onto a 12% non-denaturing polyacrylamide gel, and use 100 μV for 10 minutes at 1 V TBE buffer, then 200 V. Electrophoresis was performed for 30 minutes.
(4) 40 minutes after the start of electrophoresis, remove the gel from the gel plate and add 200 μL of staining solution A (1.0 mg/mL Stains-all (trade name) formamide solution) to the staining buffer (10% formamide, 25% 2-propanol, 65% % 15 mM Tris (pH 8.8) mixed solution (100 mL)) and shaken and agitated for 15 minutes.
(5) After 15 minutes, the gel was taken out of the staining solution A, and stained with shaking solution using staining solution B (10 mg/mL ethidium bromide aqueous solution 10 μL added to 1×TAE buffer 200 mL) for 5 minutes.
(6) After 5 minutes, the gel was taken out of the staining solution B and decolorized by shaking and stirring with 1×TBE buffer for 15 minutes.
(7) After decolorization, the gel was scanned using Typhoon 8600 (GE Healthcare) to obtain a gel image. The filters used were a 580-640 band pass filter (for Stains-all and ethidium bromide detection) and a 640-700 band pass filter (for Cy5 detection).
(8) The fluorescence image of stained DNA (green) and the fluorescence image of L1Cy5-7OTD (red) were merged using ImageJ to analyze whether L1Cy5-7OTD was bound to DNA.
(9) As a result, it was confirmed that all 97 sequences analyzed were bound to L1Cy5-7OTD.
５． CDスペクトル解析
さらに以下の通り、CDスペクトル解析を行い、四重らせん（G-quadruplex）構造の種類を調べた。
5. CD spectrum analysis Furthermore, the CD spectrum analysis was performed as follows, and the kind of quadruplex (G-quadruplex) structure was investigated.
(1) 200μM L1Cy5-7OTDを50mM Tris-HCl (pH7.5)、100mM KClを用いて調製した。
(2) (1)で調製した200μM L1Cy5-7OTD 1μLに50mM Tris-HCl (pH7.5)、100mM KCl bufferを97μL、種々の50μM DNA 2.0μLを加え、これらをCDスペクトル測定サンプルとした。また、リガンド非存在化の場合と比較するため50mM Tris-HCl (pH7.5)、100mM KCl buffer 98μLに種々の50μM DNA 2.0μLを加えたものも別途調製した。
(3) (2)で調製したCDスペクトル測定サンプル全量を、石英セル (Agilent、Microcell 50μL 10mM Path UV) に入れ、J-720 (JASCO) を用いて測定した。測定条件は、25℃において以下のように設定した。
(1) 200 μM L1Cy5-7OTD was prepared using 50 mM Tris-HCl (pH 7.5) and 100 mM KCl.
(2) To 200 μM L1Cy5-7OTD 1 μL prepared in (1), 50 mM Tris-HCl (pH 7.5), 100 mM KCl buffer (97 μL) and various 50 μM DNA 2.0 μL were added, and these were used as CD spectrum measurement samples. In addition, in order to compare with the case of no ligand, 50 mM Tris-HCl (pH 7.5) and 98 mM of 100 mM KCl buffer were also separately prepared by adding 2.0 μL of various 50 μM DNA.
(3) The total amount of the CD spectrum measurement sample prepared in (2) was placed in a quartz cell (Agilent, Microcell 50 μL 10 mM Path UV) and measured using J-720 (JASCO). The measurement conditions were set as follows at 25°C.
(4) 測定したスペクトルを、スペクトルマネージャーを用いてtxt.ファイルに変換し、グラフを作成した。
(5)以下の基準をもとに解析したDNAが3種類のG4構造（parallel type、anti-parallel type、hybrid type）のうち、どの構造を形成するか検討した。
(4) The measured spectrum was converted into a txt.file using a spectrum manager to create a graph.
(5) We examined which structure among the three types of G4 structures (parallel type, anti-parallel type, hybrid type) the DNA analyzed based on the following criteria.
(6) その結果、L1Cy5-7OTD非存在下において、#60_SP130_CGI、#70_NCOR2_CGIはanti-parallel type G-quadruplex構造を形成し、#11_SMRD1_CGI、#21_SPAS2_CGI、#27_BAI1_CGI、#30_BRM1L_CGI、#38_KDM5B_CGI、#48_MED4_CGI、#51_UBP11_CGI、#94_EDC3_CGIはhybrid type G-quadruplex構造を形成し、残り87配列はすべてparallel type G-quadruplex構造を形成することが示された（添付ファイル名：120424_ＣＤスペクトル.pptx）。
(7)また、L1Cy5-7OTD存在下で構造が変わるDNA配列として、#60_SP13_CGI(anti-parallel typeからhybrid type)、#11_SMRD1_CGI（hybrid typeからparallel type）、#48_MED4_CGI（hybrid typeからparallel type）、#45_DLX1_CGI（parallel typeからhybrid type）に変化する配列を同定している。
(6) As a result, in the absence of L1Cy5-7OTD, #60_SP130_CGI and #70_NCOR2_CGI form an anti-parallel type G-quadruplex structure, and #11_SMRD1_CGI, #21_SPAS2_CGI, #27_BAI1_CGI, #30_BRM1L_CGI, #38_CGI, #38_KGI, #38_CGI, #38_CGI, It was shown that #51_UBP11_CGI and #94_EDC3_CGI form a hybrid type G-quadruplex structure, and the remaining 87 sequences all form a parallel type G-quadruplex structure (attached file name: 120424_CD spectrum.pptx).
(7) Also, as a DNA sequence whose structure changes in the presence of L1Cy5-7OTD, #60_SP13_CGI (anti-parallel type to hybrid type), #11_SMRD1_CGI (hybrid type to parallel type), #48_MED4_CGI (hybrid type to parallel type), #45_DLX1_CGI (parallel type to hybrid type) is identified.
実施例３ ヒトゲノムＤＮＡ解析
(1) UCSC genome browser(カリフォルニア大学サンタクルーズ校により提供されているデータベース（http://genome.ucsc.edu/cgi-bin/hgTracks?org=human）からすべてのヒトCpG island領域(28691箇所)の塩基番号を取得した。塩基番号はすべてhg19(UCSCヒトゲノム１９)に対応している。
(2) 28691箇所のCpG island領域の上流90bp、下流90bpを含む領域に対してagilent e-array（商品名）を用いてタイリングアレイを作製した。(Probe length: 60-mer、space:26-bp、probe #: 962646)
(3) 終濃度10 nML1Cy5-7OTDをBuffer A (50mM Tris-HCl (pH7.5)、100mM KCl、1×Hi-RPM Hybridization Buffer (Agilent))を用いて調製した。
(4) ガスケットスライド（Agilent）に(1)で調製した10 nM L1Cy5-7OTDを490μL加え、そこに作製したヒトタイリングアレイを乗せ、ハイブリチャンバーにセットした。
(5) ハイブリチャンバーにセットしたアレイをハイブリゼーションオーブン(Agilent)にセットし、25℃、1時間、4 rpmでインキュベートした。
(6) 1時間後、ハイブリチャンバーを解体し、アレイをBuffer B (50mM Tris-HCl (pH7.5)、100mM KCl)中で5分間、25℃で洗浄した。
(7) 洗浄後、アレイをAgilent DNA Microarray scanner with surescan High resolution thchnology (Agilent)を用いてスキャンし、蛍光画像を得た。
(8) 得られた蛍光画像から、各プローブの蛍光強度(rProcessedSignal)及び塩基番号をAgilent Feature Extractionを用いて抽出した。
(9) L1Cy5-7OTDが結合しているプローブの蛍光強度の閾値を5360とし、5360以上の蛍光強度を示したプローブをMicrosoft Excel（商品名）により抽出し、さらにそれらの内、グアニンの2連続配列を4箇所以上含む配列のみ抽出した。その結果、3594プローブが抽出された。
(11) ヒトゲノムにおいて、得られ3594プローブ配列上流5kbp、下流5kbp中に存在する遺伝子をUCSC genome browerから抽出し、各プローブ近傍に存在する遺伝子をリストアップし、近傍に遺伝子が存在する2018プローブのみリストアップした（上記表１）。
Example 3 Human genomic DNA analysis
(1) UCSC genome browser (all human CpG island regions from the database (http://genome.ucsc.edu/cgi-bin/hgTracks?org=human) provided by the University of California, Santa Cruz (28691)) The base numbers were obtained, all of which correspond to hg19 (UCSC human genome 19).
(2) A tiling array was prepared using the Agilent e-array (trade name) for the region containing 90bp upstream and 90bp downstream of the 28691 CpG island region. (Probe length: 60-mer, space:26-bp, probe #: 962646)
(3) A final concentration of 10 nM L1Cy5-7OTD was prepared using Buffer A (50 mM Tris-HCl (pH 7.5), 100 mM KCl, 1×Hi-RPM Hybridization Buffer (Agilent)).
(4) 490 μL of 10 nM L1Cy5-7OTD prepared in (1) was added to a gasket slide (Agilent), and the prepared human tiling array was placed on the gasket slide and set in the hybrid chamber.
(5) The array set in the hybrid chamber was set in a hybridization oven (Agilent) and incubated at 25° C. for 1 hour at 4 rpm.
(6) After 1 hour, the hybrid chamber was disassembled, and the array was washed in Buffer B (50 mM Tris-HCl (pH 7.5), 100 mM KCl) for 5 minutes at 25°C.
(7) After washing, the array was scanned using Agilent DNA Microarray scanner with surescan High resolution thchnology (Agilent) to obtain a fluorescence image.
(8) The fluorescence intensity (rProcessedSignal) and base number of each probe were extracted from the obtained fluorescence image using Agilent Feature Extraction.
(9) The threshold of the fluorescence intensity of the probe to which L1Cy5-7OTD is bound is set to 5360, and the probe showing a fluorescence intensity of 5360 or more is extracted by Microsoft Excel (trade name), and further, guanine is continuously extracted in two of them. Only sequences containing 4 or more sequences were extracted. As a result, 3594 probe was extracted.
(11) In the human genome, the genes present in the obtained 3594 probe sequence upstream 5 kbp and downstream 5 kbp were extracted from the UCSC genome brower, and the genes existing in the vicinity of each probe were listed up. Listed (Table 1 above).
実施例４ CDスペクトル解析（その２）
(1) 終濃度2.0μM L1Cy5-7OTDを50mM Tris-HCl(pH7.5)、100mM KClを用いてサンプルを調製した。
(2) (1)で調製した2.0μM L1Cy5-7OTD 98μLに、50μM DNA 2.0μLを加え、これをCDスペクトル測定サンプルAとした。また、同様に50mM Tris-HCl (pH7.5)、100mM KClに、50μM DNA 2.0μLを加え、これをCDスペクトル測定サンプルBとした。
(3) (2)で調製した二種のCDスペクトル測定サンプル全量を、石英セル (Agilent、Microcell 50μL 10mM Path UV) に入れ、J-720 (JASCO) を用いて測定した。測定条件は、25℃において以下のように設定した。
Example 4 CD spectrum analysis (2)
(1) A sample was prepared by using a final concentration of 2.0 μM L1Cy5-7OTD in 50 mM Tris-HCl (pH 7.5) and 100 mM KCl.
(2) 2.0 μL of 50 μM DNA was added to 98 μL of 2.0 μM L1Cy5-7OTD prepared in (1), and this was designated as CD spectrum measurement sample A. Similarly, 2.0 μL of 50 μM DNA was added to 50 mM Tris-HCl (pH 7.5) and 100 mM KCl to obtain a CD spectrum measurement sample B.
(3) The total amount of the two types of CD spectrum measurement samples prepared in (2) was placed in a quartz cell (Agilent, Microcell 50 μL 10 mM Path UV) and measured using J-720 (JASCO). The measurement conditions were set as follows at 25°C.
(4) 測定したスペクトルを、スペクトルマネージャーを用いてtxt.ファイルに変換した。
(5) txtファイルのデータを基に、グラフを作成し、スペクトルを解析した。
(6) 上記表１１の基準をもとに解析したDNAが3種類のG4構造（parallel type、anti-parallel type、hybrid type）のうち、どの構造を形成するか検討した。
(7) その結果、L1Cy5-7OTD非存在下において、#60_SP130_CGIはanti-parallel type G-quadruplex構造を形成し、#13_DELE_CGI、 #95_CDC6_CGIはparallel type G-quadruplex構造を形成することが示された。
(8) また、L1Cy5-7OTD存在下では、#60_SP13_CGI、#13_DELE_CGI、#95_CDC6_CGIはいずれもhybrid typeのG-quadruplex構造に構造が変化することが示された。
(4) The measured spectrum was converted into a txt. file using the spectrum manager.
(5) A graph was created based on the data in the txt file, and the spectrum was analyzed.
(6) It was examined which of the three types of G4 structures (parallel type, anti-parallel type, hybrid type) the DNA analyzed based on the criteria in Table 11 above forms.
(7) As a result, it was shown that #60_SP130_CGI formed an anti-parallel type G-quadruplex structure, and #13_DELE_CGI and #95_CDC6_CGI formed a parallel type G-quadruplex structure in the absence of L1Cy5-7OTD.
(8) In addition, in the presence of L1Cy5-7OTD, #60_SP13_CGI, #13_DELE_CGI, and #95_CDC6_CGI were all shown to change into a hybrid type G-quadruplex structure.
実施例５ DMSフットプリント
(1) 50mM Tris-HCl (pH7.5)を用いてDNAを終濃度5.0μMに希釈しサンプルAを100μL得た。同様に、50mM Tris-HCl (pH7.5)、100mM KClを用いてサンプルBを、50mM Tris-HCl (pH7.5)、100mM KCl、L1Cy5-7OTD 50μLを用いてサンプルCをそれぞれ調製した。
(2) (1)で調製したサンプルをそれぞれ100μLずつ 1.5mLチューブに分注した。
(3) (2)を95℃で3分間静置した後、徐々に室温まで冷却した。
(4) ジメチル硫酸 (DMS) をエタノールで希釈することにより、 5.0% DMSを120μL調整した。
(5) 3M 酢酸ナトリウム緩衝液 (pH7.0) 50μL、2-メルカプトエタノール42μL、100 mg/mL tRNA 2.0μL、滅菌水18μLを混合することで反応停止液を調整した。
(6) それぞれのサンプルに対して、室温にて(4)で調整した5% DMS 10μLを加え5分間静置することでDNAのメチル化反応を行った。
(7) (6)に(5)で調製した反応停止液10μL及びエタノールを 300μL加え、反応を停止し、-80℃にて30分間静置した。
(8) (7)を4℃、15,000 rpmの条件で30分間遠心分離した。
(9) (8)の上清を除去し、3M 酢酸ナトリウム緩衝液10μL、滅菌水100μL、エタノール250μLを加えた後、再び4℃ 、15,000 rpmの条件で30分間遠心分離した。
(10) (9)の上清を取り除き、70％エタノール800μLを加えた後、4℃ 、15,000 rpmの条件で5分間遠心分離した。この操作を2回繰り返した。
(11) (10)を1.5mLチューブの蓋を開け、大気中、95℃で乾燥させた。
(12) 99%ピペリジン 110μLをDW 990μLで希釈することにより10%ピペリジン1.1mLを調製した。
(13) (12)で調製した10%ピペリジン100μLを(11)に加え、95℃で30分間静置した。
(14) (13)で調製したサンプルを凍結乾燥することで、10%ピペリジンを留去するとともに、サンプルを乾燥した固体とした。
(15) ホルムアミド788μL、0.5 M エチレンジアミン四酢酸ナトリウム (pH 8.0) 40μL、滅菌水172μLを混合することで電気泳動用の緩衝液を1mL調製した。
(16) (15)で調製した緩衝液を(14)で乾燥させたDNAサンプルそれぞれに5.0μLずつ加え、95℃で3分間静置し、その後氷上で冷却した。
(17) 7Mウレアを含む20%ポリアクリルアミドゲルを用いて、(16)で調製したサンプル1μLを電気泳動した。1000 V で10分間電気泳動した後、2500 V で100分間電気泳動した。
(18) 泳動後、ゲルをTyphoon 8600 (GE Healthcare) を用いてスキャンすることで画像を得た。なお、フィルターは526 short pass filter（商品名）を用いた。
(19) 得られた画像を、ImageJ（商品名）を用いて解析した。
(20) その結果、KCl非存在下においては、DMSでメチル化されることにより、全てのグアニン部分でDNAが切断された。一方、KCl存在下においては、一部切断からの保護が認められた。また、KCl、L1Cy5-7OTD共存下においても、同様の傾向が認められたが、一部切断からの保護のパターンは異なるものも存在した。これは、G-quadruplex中のG-quartet形成によりグアニンが水素結合を形成したことで、グアニンがメチル化反応から保護されていることを示している。また、L1Cy5-7OTD存在下においてメチル化反応からの保護のパターンが異なる配列については、KClのみの時に比べ、異なる構造のG-quadruplexへと折りたたまれていることが示された。
Example 5 DMS footprint
(1) DNA was diluted to a final concentration of 5.0 μM with 50 mM Tris-HCl (pH 7.5) to obtain 100 μL of sample A. Similarly, sample B was prepared using 50 mM Tris-HCl (pH 7.5) and 100 mM KCl, and sample C was prepared using 50 mM Tris-HCl (pH 7.5), 100 mM KCl and 50 μL of L1Cy5-7OTD.
(2) 100 μL of each of the samples prepared in (1) was dispensed into a 1.5 mL tube.
(3) After allowing (2) to stand at 95°C for 3 minutes, it was gradually cooled to room temperature.
(4) 120 μL of 5.0% DMS was prepared by diluting dimethylsulfate (DMS) with ethanol.
(5) A reaction stop solution was prepared by mixing 50 μL of 3M sodium acetate buffer (pH 7.0), 42 μL of 2-mercaptoethanol, 2.0 μL of 100 mg/mL tRNA, and 18 μL of sterilized water.
(6) To each sample, 10 μL of 5% DMS prepared in (4) was added at room temperature, and the mixture was allowed to stand for 5 minutes to perform DNA methylation reaction.
(7) 10 μL of the reaction stop solution prepared in (5) and 300 μL of ethanol were added to (6) to stop the reaction, and the mixture was allowed to stand at −80° C. for 30 minutes.
(8) (7) was centrifuged at 4°C and 15,000 rpm for 30 minutes.
(9) The supernatant of (8) was removed, 10 μL of 3M sodium acetate buffer, 100 μL of sterilized water, and 250 μL of ethanol were added, and then the mixture was centrifuged again at 4° C. and 15,000 rpm for 30 minutes.
(10) The supernatant of (9) was removed, 800 μL of 70% ethanol was added, and then the mixture was centrifuged at 4° C. and 15,000 rpm for 5 minutes. This operation was repeated twice.
(11) The lid of the 1.5 mL tube of (10) was opened and dried at 95° C. in the atmosphere.
(12) 1.1% of 10% piperidine was prepared by diluting 99 μL of 99% piperidine with 990 μL of DW.
(13) 100 μL of 10% piperidine prepared in (12) was added to (11), and the mixture was allowed to stand at 95° C. for 30 minutes.
(14) By freeze-drying the sample prepared in (13), 10% piperidine was distilled off and the sample was made into a dried solid.
(15) Formamide 788 μL, 0.5 M sodium ethylenediaminetetraacetate (pH 8.0) 40 μL, and sterilized water 172 μL were mixed to prepare 1 mL of a buffer solution for electrophoresis.
(16) The buffer solution prepared in (15) was added in an amount of 5.0 μL to each of the DNA samples dried in (14), allowed to stand at 95° C. for 3 minutes, and then cooled on ice.
(17) 1 μL of the sample prepared in (16) was electrophoresed using a 20% polyacrylamide gel containing 7M urea. After electrophoresis at 1000 V for 10 minutes, electrophoresis was performed at 2500 V for 100 minutes.
(18) After electrophoresis, the gel was scanned with Typhoon 8600 (GE Healthcare) to obtain an image. The filter used was 526 short pass filter (trade name).
(19) The obtained image was analyzed using ImageJ (trade name).
(20) As a result, in the absence of KCl, DNA was cleaved at all guanine moieties by being methylated by DMS. On the other hand, in the presence of KCl, protection from partial cleavage was observed. The same tendency was observed even in the coexistence of KCl and L1Cy5-7OTD, but some patterns had different protection patterns from partial cleavage. This indicates that guanine is protected from the methylation reaction by forming a hydrogen bond in guanine by forming G-quartet in G-quadruplex. In addition, it was shown that sequences with different patterns of protection from methylation reaction in the presence of L1Cy5-7OTD were folded into G-quadruplexes with different structures compared to KCl alone.
実施例６ ルシフェラーゼレポーターアッセイ（その２）
(1) C57BL/6マウスゲノムDNAを鋳型として、Dele、Sap130、Cdc6 CpG island (CGI)を以下の条件でPCR増幅した。
Example 6 Luciferase Reporter Assay (Part 2)
(1) Using C57BL/6 mouse genomic DNA as a template, Dele, Sap130, and Cdc6 CpG island (CGI) were PCR-amplified under the following conditions.
(2) PCR産物をWizard（登録商標） SV Gel and PCR Clean-Up System(Promega)を用いて精製し、以下の組成でPCR産物とSfiI (NEB)を混合し、50℃、2時間インキュベートすることによりPCR産物を切断した。 (2) The PCR product was purified using Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega), the PCR product and SfiI (NEB) were mixed in the following composition, and the mixture was incubated at 50°C for 2 hours. The PCR product was cleaved by this.
(3) (2)と同様にホタルルシフェラーゼ遺伝子を持つpGL4.23 vector (promega)をSfiIで切断した。組成は以下に示す。 (3) As in (2), pGL4.23 vector (promega) containing the firefly luciferase gene was digested with SfiI. The composition is shown below.
(4) (2)、(3)で切断したPCR産物及びpGL4 vectorを1%アガロースを用いて電気泳動し、目的のバンドを切り出し、Wizard（商品名） SV Gel and PCR Clean-Up System(Promega)を用いて精製した。
(5) (4)で調製したpGL4 vector (18 fmol)、PCR産物(54 fmol)を混合し、Ligation high Ver.2(LGK-201,TOYOBO)を用いて5min 室温でライゲーションした。組成は以下に示す。
(4) The PCR product cleaved in (2) and (3) and the pGL4 vector were electrophoresed using 1% agarose, the desired band was cut out, and the Wizard (trade name) SV Gel and PCR Clean-Up System (Promega ) Was used for purification.
(5) The pGL4 vector (18 fmol) prepared in (4) and the PCR product (54 fmol) were mixed and ligated with Ligation high Ver.2 (LGK-201, TOYOBO) at room temperature for 5 min. The composition is shown below.
(6) (5)で得られたライゲーションサンプルを7μL用い、(200μLに対して3.4μLのβ-メルカプトエタノールを加え氷上で10分インキュベートした)70μLのJM110コンピテントセル(Agilent)に加え、30分氷上でインキュベートした。30分後、ヒートショック(42℃45秒インキュベート後、氷上で2分間インキュベート)を行い、得られた菌体をアンピシリンを含むLBプレートにプレーティングし、37℃で一晩培養した。
(7) 得られたコロニーをピックアップし、3mLのLB培地で37℃で一晩培養した。
(8) 得られた菌体からPureYield（商品名）Plasmid Miniprep System(promega)を用いてプラスミドを調製した。
(9) 得られたプラスミドをシークエンスし、ルシフェラーゼ遺伝子上流の目的の位置に標的CpG islandがクローニングされていることを確認した。
(10)600 fmolの各プラスミドとコントロールベクターである12 fmolのウミシイタケルシフェラーゼ遺伝子発現vector pGL4.74(promega)を混合し、Opti-MEM(Invitrogen)を用いて300μLに調整し、6μL Lipofectoamine2000 (Invitrogen)と294μL Opti-MEMの混合液300μLを加え、20分間室温でインキュベートした。
(11)前日に5×103 cell/wellで24wellプレートに播種したHeLa細胞の培地を400μLのOpti-MEMに交換し、そこに(10)で調製したプラスミド混合溶液を100μLずつ6wellに加え、37℃、5% CO2条件下で4時間インキュベートした。
(12) 4時間後、1μM 7OTDを含む培地又は7OTDを含まない培地に交換し、20時間37℃、5% CO2条件下で培養した。
(13) 20時間後、Dual-Luciferase reporter Assay system (promega)を用いて、ホタルルシフェラーゼ及びウミシイタケルシフェラーゼの発光量を定量した。発光量はARVOMX 1420 Multilabel counter(Perkin Elmer)を用いて定量した。
(14) それぞれのホタルルシフェラーゼの発現量はコントロールベクターから発現したウミシイタケルシフェラーゼの発現量で標準化した。
(15)その結果、プロモーターをクローニングしていないベクターをトランスフェクションした場合と比較すると、Dele、Sap130、Cdc6 CGIをクローニングした場合のホタルルシフェラーゼの発現量はそれぞれ46.4、365、157倍になった(下記表１９)。つまり、Dele、Sap130、Cdc6 はHeLa細胞中でプロモーター活性もしくはエンハンサー活性を持つことが示された。1μM Gqリガンドを培地に加えるとSap130 CGIをクローニングしたvectorではホタルルシフェラーゼの発現量の減少が観察された。つまり、Sap130 CGIに7OTDが結合することにより転写活性が減少することが示唆された。
(6) Using 7 μL of the ligation sample obtained in (5), added to 70 μL of JM110 competent cells (Agilent) (3.4 μL of β-mercaptoethanol was added to 200 μL and incubated for 10 minutes), 30 Incubated on ice cubes. After 30 minutes, heat shock (incubation at 42° C. for 45 seconds and then on ice for 2 minutes) was performed, and the obtained bacterial cells were plated on an LB plate containing ampicillin and cultured overnight at 37° C.
(7) The obtained colonies were picked up and cultured in 3 mL of LB medium at 37°C overnight.
(8) A plasmid was prepared from the obtained cells using PureYield (trade name) Plasmid Miniprep System (promega).
(9) The obtained plasmid was sequenced, and it was confirmed that the target CpG island was cloned at the target position upstream of the luciferase gene.
(10) 600 fmol of each plasmid and 12 fmol of Renilla luciferase gene expression vector pGL4.74 (promega), which is a control vector, were mixed, adjusted to 300 μL using Opti-MEM (Invitrogen), and 6 μL Lipofectoamine2000 (Invitrogen). ) And 294 μL Opti-MEM mixed solution (300 μL) was added, and the mixture was incubated at room temperature for 20 minutes.
(11) The medium of HeLa cells seeded on a 24-well plate at 5×10 3 cells/well on the previous day was replaced with 400 μL of Opti-MEM, and the plasmid mixed solution prepared in (10) was added to 6 wells of 100 μL each. Incubated at 37°C, 5% CO 2 for 4 hours.
(12) After 4 hours, the medium was replaced with a medium containing 1 μM 7OTD or a medium not containing 7OTD, and cultured for 20 hours at 37° C. under 5% CO 2 condition.
(13) After 20 hours, the amount of luminescence of firefly luciferase and Renilla luciferase was quantified using the Dual-Luciferase reporter Assay system (promega). Luminescence was quantified using an ARVO MX 1420 Multilabel counter (Perkin Elmer).
(14) The expression level of each firefly luciferase was standardized by the expression level of Renilla luciferase expressed from the control vector.
(15) As a result, the expression levels of firefly luciferase in the case of cloning Dele, Sap130, and Cdc6 CGI were 46.4, 365, and 157 times, respectively, as compared with the case of transfecting a vector in which the promoter was not cloned (respectively). Table 19 below). That is, Dele, Sap130, and Cdc6 were shown to have promoter activity or enhancer activity in HeLa cells. When 1 μM Gq ligand was added to the medium, a decrease in the expression level of firefly luciferase was observed in the Sap130 CGI cloned vector. In other words, it was suggested that the binding of 7OTD to Sap130 CGI reduces the transcriptional activity.
Claims (13)
Ｒ１は、単結合、炭素数１〜１２のアルキレン基、炭素数１〜１２のアルケニレン基又は炭素数１〜１２のアルキニレン基を表す。
Ｒ２、Ｒ３、Ｒ４、Ｒ５、Ｒ６及びＲ７は、それぞれ独立に、水素原子、アミノ基若しくはグアニジノ基を有していてもよい炭素数１〜１２のアルキル基、アミノ基、グアニジノ基、アミノ基若しくはグアニジノ基を有していてもよい炭素数７〜１２のアラルキル基、アミノ基若しくはグアニジノ基を有していてもよいフェニル基、炭素数２〜６の複素環基又はハロゲン原子を表す。
Ｒ8は単結合、炭素数１〜１２のアルキレン基、炭素数１〜１２のアルケニレン基又は炭素数１〜１２のアルキニレン基を表す。
Ｑはシアニン蛍光色素を表す。 A quadruplex DNA detection probe having a chemical structure represented by the general formula [I].
R 1 represents a single bond, an alkylene group having 1 to 12 carbon atoms, an alkenylene group having 1 to 12 carbon atoms, or an alkynylene group having 1 to 12 carbon atoms.
R 2 , R 3 , R 4 , R 5 , R 6 and R 7 are each independently a hydrogen atom, an alkyl group having 1 to 12 carbon atoms which may have an amino group or a guanidino group, an amino group, A guanidino group, an aralkyl group having 7 to 12 carbon atoms which may have an amino group or a guanidino group, a phenyl group which may have an amino group or a guanidino group, a heterocyclic group having 2 to 6 carbon atoms, or a halogen. Represents an atom.
R 8 represents a single bond, an alkylene group having 1 to 12 carbon atoms, an alkenylene group having 1 to 12 carbon atoms, or an alkynylene group having 1 to 12 carbon atoms.
Q represents a cyanine fluorescent dye.
各円弧は、Ｎをヘテロ原子として含む環状構造（縮合環である場合を包含する）を示す。 The probe according to claim 2, wherein the cyanine fluorescent dye is represented by the following general formula [II].
Each arc represents a cyclic structure containing N as a hetero atom (including the case of a condensed ring).
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