JP2016056136A - Vascular endothelial growth factor binding nucleic acid aptamer and use thereof - Google Patents
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Abstract
Description
本発明は、新規な血管内皮細胞増殖因子（VEGF）結合性核酸アプタマー及びその利用に関する。 The present invention relates to a novel vascular endothelial growth factor (VEGF) -binding nucleic acid aptamer and use thereof.
VEGFは血管の内皮細胞に存在する受容体に結合し、内皮細胞の増殖を促すことで、血管新生を促進するタンパク質である。VEGFを標的とした薬剤は、血管新生を阻害して腫瘍の成長を抑制する抗癌剤や加齢黄斑変性症の治療薬として実用化されている。また、固形腫瘍患者の血清中でのVEGFの濃度上昇が報告されている。このため、VEGFに結合する物質が得られれば、血管新生を伴うさまざまな疾患の診断や治療を行う上で有効である。 VEGF is a protein that promotes angiogenesis by binding to receptors present on vascular endothelial cells and promoting proliferation of the endothelial cells. Drugs targeting VEGF have been put into practical use as anticancer agents that inhibit angiogenesis and suppress tumor growth and as therapeutic agents for age-related macular degeneration. In addition, an increase in the concentration of VEGF in the serum of patients with solid tumors has been reported. Therefore, obtaining a substance that binds to VEGF is effective in diagnosing and treating various diseases associated with angiogenesis.
VEGFに結合するアプタマーが報告されている（特許文献１）。しかし、これらは天然の塩基（ATGC）からなる配列であり、安定性等の面で十分とは言えなかった。また、本発明者は修飾核酸を含んだ核酸アプタマーを合成し、SELEX法により、カンプトテシン類に特
異的に結合するアプタマーを取得している（特許文献２）。
An aptamer that binds to VEGF has been reported (Patent Document 1). However, these are sequences composed of natural bases (ATGC), which are not sufficient in terms of stability and the like. Further, the present inventors have synthesized a nucleic acid aptamer containing a modified nucleic acid and obtained an aptamer that specifically binds to camptothecins by the SELEX method (Patent Document 2).
本発明の目的は、VEGFを特異的に認識し結合する核酸アプタマーを提供することである。 An object of the present invention is to provide a nucleic acid aptamer that specifically recognizes and binds VEGF.
本発明者は上記課題を解決するために鋭意検討を行った。その結果、特定の修飾核酸残基を含む核酸アプタマーがVEGFに特異的に結合することを見出し、それにより、VEGFの測定やVEGFを標的とした医薬に利用できることを見出して、本発明を完成させるに至った。 The present inventor has intensively studied to solve the above problems. As a result, it has been found that a nucleic acid aptamer containing a specific modified nucleic acid residue specifically binds to VEGF, thereby finding that it can be used for measurement of VEGF or a drug targeting VEGF, and completes the present invention. It came to.
すなわち、本発明は以下の発明を提供する。
［１］下記（I）の修飾核酸の残基を含む核酸アプタマーを含むVEGF結合剤。
［２］核酸アプタマーが一本鎖ＤＮＡである、［１］に記載のVEGF結合剤。
［３］核酸アプタマーの長さが１５〜１００塩基である、［１］または［２］に記載のVEGF結合剤。
［４］核酸アプタマーの配列が配列番号４〜２０のいずれかの塩基番号２１〜５０の配列、配列番号２１〜２３のいずれかの塩基番号２１〜４９の配列、または配列番号２４の塩基番号２１〜５１の配列（これらの配列におけるＴは下記の式（I）で表される化合物の
残基を示す）を含む、［１］〜［３］のいずれかに記載のVEGF結合剤。
［５］下記（I）の修飾核酸の残基を含む核酸アプタマーのライブラリーを用意する工程
、およびライブラリーをVEGFと反応させる工程、およびVEGFと結合した核酸アプタマーを選択して増幅する工程を含む、VEGF結合剤のセレクション方法。
That is, the present invention provides the following inventions.
[1] A VEGF binding agent comprising a nucleic acid aptamer comprising a modified nucleic acid residue of the following (I).
[2] The VEGF binding agent according to [1], wherein the nucleic acid aptamer is single-stranded DNA.
[3] The VEGF binding agent according to [1] or [2], wherein the nucleic acid aptamer has a length of 15 to 100 bases.
[4] Nucleic acid aptamer sequence of any of
[5] A step of preparing a library of nucleic acid aptamers containing residues of the modified nucleic acid of the following (I), a step of reacting the library with VEGF, and a step of selecting and amplifying the nucleic acid aptamer bound to VEGF A method for selecting a VEGF binding agent.
本発明において新たに開発した核酸アプタマーは、血液系標的であるVEGFを特異的に認識し結合することができるため、癌の診断マーカーおよび抗癌剤の分子標的として有力な
候補である。
Since the nucleic acid aptamer newly developed in the present invention can specifically recognize and bind to blood system target VEGF, it is a promising candidate as a diagnostic marker for cancer and a molecular target for anticancer agents.
以下に本発明を詳しく説明する。
本発明のVEGF結合剤は、下記（I）の修飾核酸の残基（ヌクレオチド残基）を含む核酸アプタマーである。
The VEGF binding agent of the present invention is a nucleic acid aptamer containing the following modified nucleic acid residue (nucleotide residue) (I).
（I）の修飾核酸は特開２０１３−４０１１８に記載の方法によって合成することがで
きる。
The modified nucleic acid (I) can be synthesized by the method described in JP2013-40118.
核酸アプタマーとは好ましくはDNA（ポリヌクレオチド）であり、より好ましくは一本
鎖DNA（ポリヌクレオチド）である。
核酸アプタマーの長さは、好ましくは１５〜１００塩基であり、より好ましくは２０〜６０塩基である。
核酸アプタマーはさらに修飾されたものであってもよい。例えば、核酸アプタマーの安定性を増加させるために、ホスホロチオエートまたはホスホロジチオエート修飾することも可能である。また、蛍光物質等で標識されたものでもよい。
The nucleic acid aptamer is preferably DNA (polynucleotide), more preferably single-stranded DNA (polynucleotide).
The length of the nucleic acid aptamer is preferably 15 to 100 bases, more preferably 20 to 60 bases.
The nucleic acid aptamer may be further modified. For example, phosphorothioate or phosphorodithioate modifications can be made to increase the stability of the nucleic acid aptamer. Further, it may be labeled with a fluorescent substance or the like.
（I）の修飾核酸の残基を含む核酸アプタマーとは、アデニン（A）、チミン（T）、グ
アニン（G）、シトシン（C）の４種類の天然の塩基のうちの１種類以上とともに、（I）
の修飾核酸の残基を含む核酸アプタマーを意味するが、（I）の修飾核酸はチミジン5’-
三リン酸（dTTP）の誘導体であるため、チミン（T）の代わりに（I）の修飾核酸の残基が含まれる核酸アプタマーであることは好ましい。すなわち、アデニン（A）、グアニン（G）、シトシン（C）の３種類の天然の塩基のうちの１種類以上とともに、（I）の修飾核酸の残基を含む核酸アプタマーであることが好ましい。核酸アプタマー全長における（I）
の修飾核酸の残基の割合は１０〜５０％であることが好ましい。
本発明の核酸アプタマーは、（I）の修飾核酸残基に加えて、他の修飾核酸残基が含ま
れてもよい。
The nucleic acid aptamer containing a residue of the modified nucleic acid (I) is one or more of four natural bases of adenine (A), thymine (T), guanine (G), and cytosine (C), (I)
Means a nucleic acid aptamer comprising residues of the modified nucleic acid of (I), wherein the modified nucleic acid of (I) is thymidine 5′-
Since it is a derivative of triphosphate (dTTP), it is preferably a nucleic acid aptamer containing the residue of the modified nucleic acid (I) instead of thymine (T). That is, it is preferably a nucleic acid aptamer containing a residue of the modified nucleic acid (I) together with one or more of three natural bases of adenine (A), guanine (G), and cytosine (C). Nucleic acid aptamer full length (I)
The percentage of residues in the modified nucleic acid is preferably 10 to 50%.
The nucleic acid aptamer of the present invention may contain other modified nucleic acid residues in addition to the modified nucleic acid residue of (I).
本発明の核酸アプタマーは、DNA合成機などを使用して得ることができる。例えば、DNA合成機に、アデニン（A）、グアニン（G）、シトシン（C）と、（I）の修飾核酸の3’-ホスホロアミダイトを用いて目的の配列の核酸アプタマーを合成することができる。 The nucleic acid aptamer of the present invention can be obtained using a DNA synthesizer or the like. For example, a DNA synthesizer can synthesize a nucleic acid aptamer of the target sequence using adenine (A), guanine (G), cytosine (C), and 3'-phosphoramidite of the modified nucleic acid (I). it can.
本発明の核酸アプタマーの具体的な態様としては、VEGF結合能を有する限り特に限定はされないが、核酸アプタマーの配列が配列番号４〜２０のいずれかの塩基番号２１〜５０の配列、配列番号２１〜２３のいずれかの塩基番号２１〜４９の配列、または配列番号２４の塩基番号２１〜５１の配列を含む核酸アプタマーが挙げられる。これらの配列において、Tは上記（I）の修飾核酸残基を意味する。
なお、VEGFに結合する能力が維持される限り、これらの配列において１〜数個、例えば、１、２または３個の塩基が置換、欠失、挿入等されてよい。また、VEGF結合能が維持される限り、５’側および／または３’側に任意の長さの任意の配列が付加されてよい。
The nucleic acid aptamer of the present invention is not particularly limited as long as it has VEGF binding ability, but the sequence of the nucleic acid aptamer is any one of
As long as the ability to bind to VEGF is maintained, 1 to several, for example, 1, 2 or 3 bases may be substituted, deleted, inserted or the like in these sequences. Moreover, as long as VEGF binding ability is maintained, arbitrary sequences of arbitrary length may be added to 5 'side and / or 3' side.
VEGF結合能としては、ヒトVEGFとKd（解離定数）が１０ｎＭ以下の親和性を示すことが好ましい。 As VEGF binding ability, it is preferable that human VEGF and Kd (dissociation constant) have an affinity of 10 nM or less.
本発明のVEGF結合剤は、アデニン（A）、チミン（T）、グアニン（G）、シトシン（C）の４種類の天然の塩基のうちの１種類以上とともに、（I）の修飾核酸の残基を含む核酸
アプタマーのライブラリーを用意し、それとVEGFを接触させ、VEGFと結合する核酸アプタマーを選択し、増幅することを繰り返すことによって核酸アプタマーのライブラリーからセレクションすることもできる。
The VEGF binding agent of the present invention comprises one or more of four natural bases of adenine (A), thymine (T), guanine (G), and cytosine (C), and the remaining modified nucleic acid of (I). It is also possible to select from a library of nucleic acid aptamers by preparing a library of nucleic acid aptamers containing a group, contacting it with VEGF, selecting a nucleic acid aptamer that binds to VEGF, and repeating amplification.
本発明のVEGF結合剤は、VEGF検出のための分子プローブとして使用することができる。例えば、核酸アプタマーを蛍光色素などで標識し、VEGFの体内動態などをトレースすることができる。VEGFは癌や、糖尿病性網膜症などの異常血管新生、関節リウマチなどの炎症性疾患などで高発現することが知られているので、本発明のVEGF結合剤はこれらの疾患の診断試薬として使用することができる。 The VEGF binding agent of the present invention can be used as a molecular probe for VEGF detection. For example, a nucleic acid aptamer can be labeled with a fluorescent dye or the like to trace the pharmacokinetics of VEGF. Since VEGF is known to be highly expressed in cancer, abnormal angiogenesis such as diabetic retinopathy, and inflammatory diseases such as rheumatoid arthritis, the VEGF binding agent of the present invention is used as a diagnostic reagent for these diseases can do.
また、腫瘍組織では血管新生が亢進しており、腫瘍組織にはVEGFが集積するため、本発明の核酸アプタマーは、抗癌剤のDDS（薬剤伝達システム）のために使用することができ
る。アプタマーに抗癌剤の有効成分を結合させ、投与することで、抗癌剤を腫瘍組織に効率よく送達することができる。
例えば、本発明の核酸アプタマーと特開２０１３−４０１１８に開示されたカンプトテシン類との結合に関与する核酸アプタマーを連結し、これに、カンプトテシン類を結合させることで、カンプトテシン類を腫瘍組織に到達させるための医薬を作製することができる。
In addition, angiogenesis is enhanced in tumor tissues, and VEGF is accumulated in tumor tissues. Therefore, the nucleic acid aptamer of the present invention can be used for DDS (drug delivery system) of anticancer agents. The anticancer agent can be efficiently delivered to the tumor tissue by binding and administering the active ingredient of the anticancer agent to the aptamer.
For example, a nucleic acid aptamer involved in the binding between the nucleic acid aptamer of the present invention and the camptothecins disclosed in JP2013-40118 is linked, and the camptothecins are allowed to reach the tumor tissue by binding the camptothecins thereto. Can be made.
以下に実施例を示し、本発明をさらに具体的に説明する。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。 The following examples illustrate the present invention more specifically. However, the present invention is not limited to the following examples.
核酸誘導体（I）は特開２０１３−４０１１８に記載の方法に従って合成した（以下、KS9とも呼ぶ）。 The nucleic acid derivative (I) was synthesized according to the method described in JP2013-40118 (hereinafter also referred to as KS9).
実施例１
CE-SELEXの実験方法
・用いたDNA
GOL#P1P:5’-TCG CTC GGC AGG ATC GCA AG-3’(5’リン酸化)（配列番号１）
GOL#P1F:5’-TCG CTC GGC AGG ATC GCA AG-3’(5’FITC化(6-FAM)) （配列番号１）
GOL#P2P:5’-TGC TGC CAC TGC TCC GTC CA-3’(5’リン酸化) （配列番号２）
GOL#P2H:5’-TGC TGC CAC TGC TCC GTC CA-3’（配列番号２）
GOL#T2H:5’-TGC TGC CAC TGC TCC GTC CAN NNN NNN NNN NNN NNN NNN NNN NNN
NNN NNC TTG CGA TCC TGC CGA GCG A-3’（配列番号３）
Example 1
CE-SELEX experimental method and DNA used
GOL # P1P: 5'-TCG CTC GGC AGG ATC GCA AG-3 '(5' phosphorylated) (SEQ ID NO: 1)
GOL # P1F: 5'-TCG CTC GGC AGG ATC GCA AG-3 '(5'FITC conversion (6-FAM)) (SEQ ID NO: 1)
GOL # P2P: 5'-TGC TGC CAC TGC TCC GTC CA-3 '(5' phosphorylated) (SEQ ID NO: 2)
GOL # P2H: 5'-TGC TGC CAC TGC TCC GTC CA-3 '(SEQ ID NO: 2)
GOL # T2H: 5'-TGC TGC CAC TGC TCC GTC CAN NNN NNN NNN NNN NNN NNN NNN NNN
NNN NNC TTG CGA TCC TGC CGA GCG A-3 '(SEQ ID NO: 3)
[修飾DNAライブラリーの酵素的調製]
1.5mLマイクロチューブに反応液を以下のように調製した。これを500μLマイクロチュ
ーブに100μLずつ8本に小分けし、遺伝子増幅装置にセットして以下の温度条件でPCRを行った。PCR産物の確認は10%変性ポリアクリルアミドゲル電気泳動（TBE Buffer、200V、45℃、35分）の後、外部レーザー（488nm）照射によって行った。確認後、反応液を10%変性ポリアクリルアミドゲル電気泳動（TB Buffer、300V、4℃、200-240分）により目的の70merのDNAバンドを外部レーザー（488nm）照射で検出し、ゲルから切り出した。切り出したゲルを、透析チューブを用いて透析（TB Buffer、100V、4℃、80分）を行った。抽出したDNA溶出液を遠心フィルターユニットにより脱塩し、凍結乾燥後、適量の蒸留水で溶解さ
せて濃度を測定することでライブラリーを得た。
[Enzymatic preparation of modified DNA library]
The reaction solution was prepared in a 1.5 mL microtube as follows. This was subdivided into 8 pieces of 100 μL each in a 500 μL microtube, set in a gene amplification apparatus, and subjected to PCR under the following temperature conditions. The PCR product was confirmed by 10% denaturing polyacrylamide gel electrophoresis (TBE Buffer, 200 V, 45 ° C., 35 minutes) followed by irradiation with an external laser (488 nm). After confirmation, the reaction solution was detected by external laser (488nm) irradiation with 10% denaturing polyacrylamide gel electrophoresis (TB Buffer, 300V, 4 ° C, 200-240 minutes) and cut out from the gel. . The excised gel was dialyzed (TB Buffer, 100 V, 4 ° C., 80 minutes) using a dialysis tube. The extracted DNA eluate was desalted with a centrifugal filter unit, freeze-dried, dissolved in an appropriate amount of distilled water, and the concentration was measured to obtain a library.
[NECEEMによる修飾DNAライブラリーのセレクション]
500μLマイクロチューブに上記で合成したライブラリーを緩衝液中で1μMになるように調製した。緩衝液にはTris-HCl buffer（20mM Tris-HCl（pH 7.4）、1mM MgCl2、10mM NaCl）を用いた。これを遺伝子増幅装置にセットして、94℃で0.5分熱変性し2.5hかけて25℃まで下げてアニーリングを行った。その後、標的分子であるヒトVEGFタンパク質がファイナル0.5μM(1ラウンド目のみ)もしくは0.2μM(2ラウンド目以降)になるように加えて37
℃で30分インキュベートした。ネガティブコントロールにはバッファーを加えた。この際、DNAの濃度は0.5μMとなるようにした。インキュベート後、非平衡キャピラリー電気泳動法（NECEEM）によって測定した。測定条件には下記の条件を用いた。ネガティブコントロールと比べて蛍光強度の増加がみられる部分を分取した。また、キャピラリー内壁へのDNAの吸着を確認するために、反応液をインジェクトする前にも分取している。
[Selection of modified DNA library by NECEEM]
The library synthesized above in a 500 μL microtube was prepared to 1 μM in buffer. Tris-HCl buffer (20 mM Tris-HCl (pH 7.4), 1 mM MgCl 2 , 10 mM NaCl) was used as a buffer. This was set in a gene amplification apparatus, heat denatured at 94 ° C. for 0.5 minutes, and annealed by lowering to 25 ° C. over 2.5 hours. After that, the target molecule, human VEGF protein, was added to a final 0.5 μM (only in the first round) or 0.2 μM (after the second round).
Incubated for 30 minutes at ° C. Buffer was added to the negative control. At this time, the concentration of DNA was set to 0.5 μM. After incubation, it was measured by non-equilibrium capillary electrophoresis (NECEEM). The following conditions were used as measurement conditions. The portion where the fluorescence intensity was increased compared to the negative control was collected. In addition, in order to confirm the adsorption of DNA to the inner wall of the capillary, it is also collected before injecting the reaction solution.
＜泳動条件＞
Capillary length: 80cm
Running buffer: 100mM Boric acid buffer (pH 8.4)
Temperature
Cartridge : 25℃
Sample storage : 15℃
Dynamic range: 1000RFU
Excitation wavelength: 488nm
Emission wavelength: 520nm
<Electrophoretic conditions>
Capillary length: 80cm
Running buffer: 100mM Boric acid buffer (pH 8.4)
Temperature
Cartridge: 25 ℃
Sample storage: 15 ℃
Dynamic range: 1000RFU
Excitation wavelength: 488nm
Emission wavelength: 520nm
[分取液のPCR (1st PCR)による修飾DNAライブラリー濃縮の確認]
500μLマイクロチューブに反応液を以下のように調製した。これを遺伝子増幅装置にセットし、上記セレクションで濃縮された画分を鋳型にし、以下の温度条件で1st PCRを行
った。PCR産物の確認は10％変性ポリアクリルアミドゲル電気泳動（TBE Buffer、200V、45℃、35分）の後、外部レーザー（488nm）照射によって行った。反応液を酢酸ナトリウムでエタノール沈殿し、凍結乾燥した後、残渣を200μLの蒸留水に溶解した。この溶液を2nd PCRの鋳型とした。
[Confirmation of enrichment of modified DNA library by PCR of preparative solution (1 st PCR)]
The reaction solution was prepared in a 500 μL microtube as follows. This was set in a gene amplification apparatus, a fraction enriched with the selection as a template, was subjected to 1 st PCR under the following temperature conditions. PCR products were confirmed by 10% denaturing polyacrylamide gel electrophoresis (TBE Buffer, 200 V, 45 ° C., 35 minutes) followed by irradiation with an external laser (488 nm). The reaction solution was ethanol precipitated with sodium acetate and lyophilized, and the residue was dissolved in 200 μL of distilled water. This solution was used as a template for 2 nd PCR a.
[2nd PCRおよび鋳型鎖の調製]
1.5mLマイクロチューブに反応液を以下のように調製した。これを500μLマイクロチュ
ーブに100μLずつ8本に小分けし、遺伝子増幅装置にセットして、上記で得られた1stPCR
産物を鋳型とし、以下の温度条件で対称PCRを行った。PCR産物の確認は10％変性ポリアクリルアミドゲル電気泳動（TBE Buffer、200V、45℃、35分）の後、外部レーザー（488nm
）照射によって行った。反応液を酢酸ナトリウムでエタノール沈殿し、凍結乾燥した後、残渣を80μLの蒸留水に溶解した。定法に従って、GOL#P1P伸長鎖を、λ-exonucleaseを用いて選択的に分解し、次のラウンドの修飾DNAライブラリー調製のための鋳型鎖を得た。
Preparation of 2 nd PCR and the template strand]
The reaction solution was prepared in a 1.5 mL microtube as follows. Divide this into 8 pieces of 100 μL each in a 500 μL microtube, set it in the gene amplification device, and obtain the 1 st PCR obtained above.
Using the product as a template, symmetric PCR was performed under the following temperature conditions. Confirmation of PCR products is 10% denaturing polyacrylamide gel electrophoresis (TBE Buffer, 200V, 45 ° C, 35 minutes), followed by external laser (488nm
) Performed by irradiation. The reaction solution was ethanol precipitated with sodium acetate and freeze-dried, and the residue was dissolved in 80 μL of distilled water. According to a conventional method, the GOL # P1P extended strand was selectively degraded using λ-exonuclease to obtain a template strand for the preparation of the next round of modified DNA library.
タマーを選択した。KS9を含むライブラリーから得られたVEGF結合アプタマーの配列を図
１に示す。
Kd測定の結果
上記で得られたVEGF結合アプタマーのうち、一部のアプタマーの活性評価をCE（キャピラリー電気泳動）により行った。また、キャピラリーの長さは30.2cmのものを使用し、injection時間は12secとった。他の条件は同じである。複合体平均と標準偏差は3回測定に
より算出してある。結果を図２〜図５に示した。
いずれのアプタマーもVEGFと高親和性を示すことが分かった。
Results of Kd measurement Among the VEGF-binding aptamers obtained above, the activity of some aptamers was evaluated by CE (capillary electrophoresis). A capillary with a length of 30.2 cm was used, and the injection time was 12 seconds. Other conditions are the same. Complex averages and standard deviations are calculated from three measurements. The results are shown in FIGS.
Both aptamers were found to show high affinity with VEGF.
Claims (5)
す）を含む、請求項１〜３いずれか一項に記載の血管内皮細胞増殖因子結合剤。 The sequence of the nucleic acid aptamer is any of the sequences of base numbers 21 to 50 of SEQ ID NOs: 4 to 20, the sequence of base numbers 21 to 49 of SEQ ID NOs: 21 to 23, or the base numbers of 21 to 51 of SEQ ID NO: 24 The vascular endothelial growth factor binding agent according to any one of claims 1 to 3, comprising a sequence of (T in these sequences represents a residue of the compound represented by (I)).
びライブラリーを血管内皮細胞増殖因子と反応させる工程、および血管内皮細胞増殖因子と結合した核酸アプタマーを選択して増幅する工程を含む、血管内皮細胞増殖因子結合剤のセレクション方法。
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