JP6128621B2 - マイクロベシクルに対する核酸アプタマー - Google Patents
マイクロベシクルに対する核酸アプタマー Download PDFInfo
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- JP6128621B2 JP6128621B2 JP2015551533A JP2015551533A JP6128621B2 JP 6128621 B2 JP6128621 B2 JP 6128621B2 JP 2015551533 A JP2015551533 A JP 2015551533A JP 2015551533 A JP2015551533 A JP 2015551533A JP 6128621 B2 JP6128621 B2 JP 6128621B2
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- nucleic acid
- aptamer
- microvesicle
- binding
- motif
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- C—CHEMISTRY; METALLURGY
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Description
培養液中のマイクロベシクルはSokolovaらの方法(Sokolova,V., et al., Colloids Surf B Biointerfaces, Vol.87, pp.146−150, 2011)に基づき、若干の改変を加えて調製した。10cmシャーレで培養した293T細胞の培養液(10%FBS/DMEM)を、無血清のAdvanced DMEM(ライフテクノロジーズ社製)に置換し、3日間培養した後、培養上清を回収した。回収した培養上清を0.22μmの孔径のフィルターでろ過し、200nm以上の物質を除いた。続いて、10kDaの孔径の限外ろ過フィルター(Amicon Ultra、ミリポア社製)を用いて、40mlのPBS(−)により3回バッファー交換を行った後、サンプル溶液量を約1mlに濃縮した。その後、100kDaの孔径の限外ろ過フィルター(VIVACON 500、ザルトリウス・ステディム社製)を用いて、500μlのPBS(−)により3回バッファー交換を行った。得られたサンプルを、セファロース2B(GEヘルスケア・ジャパン社製)を充填したカラムで分画し、マイクロベシクル画分を取得した。タンパク質量のプロファイルと、抗CD63抗体によるドットブロットの結果から、得られたマイクロベシクルの大半がエキソソームであることが確認された(図5)。
マイクロベシクル結合性核酸アプタマーは、SELEX法により作製した。SELEX法は、Ellingtonらの方法(Ellington,AD. and Szostak,JW., Nature, Vol.346, pp.818−822, 1990)およびGoldらの方法(Tuerk,C. and Gold,L., Science, Vol.249, pp.505−510, 1990)を参考にし、西川らの方法に基づいて行った(Murakami,K., Nishikawa,F., Noda,K., Yokoyama,T., and Nishikawa,S., Prion, Vol.2, pp.73−80, 2008)。
(Sプール)
鋳型DNA(S): 5’−gggaggtggaactgaaggaga−n55−acttcgcaatcgctctacgca−3’(配列番号1)
フォワード(Fwd)プライマー(S): 5’−tgtaatacgactcactatagggaggtggaactgaaggaga−3’(配列番号2)
リバース(Rev)プライマー(S): 5’−tgcgtagagcgattgcgaagt−3’(配列番号3)
(Kプール)
鋳型DNA(K): 5’−ggtagatacgatgga−n30−catgacgcgcagcca−3’(配列番号4)
フォワード(Fwd)プライマー(K): 5’−tgtaatacgactcactataggtagatacgatgga−3’(配列番号5)
リバース(Rev)プライマー(K): 5’−tggctgcgcgtcatg−3’(配列番号6)
nは、a,c,gまたはtを示す。Fwdプライマーは、T7 RNAポリメラーゼのプロモーター配列を含む。
上記SプールおよびKプールから得られたアプタマーの上位50配列に対して、MEME Suite(http://meme.nbcr.net/meme/)を使用してモチーフ検索を行った。
続いて、上記のモチーフMsおよびMkの二次構造予測を行った。モチーフMsを有するアプタマーである配列番号114、および、モチーフMkを有するアプタマーである配列番号159について、CentroidFoldプログラム(Nucl.Acids Res., Vol.37(Suppl.2), pp.W277−W280, 2009)により二次構造を予測した。
モチーフMsを有するアプタマー:
5’−X1−Ms−X2−3’(ここで、Msは、YRCGGHGGWRWKGGGRN(配列番号207)であり、Rは、それぞれ独立に、AまたはGであり、Yは、CまたはTもしくはUであり、Wは、それぞれ独立に、AまたはTもしくはUであり、Nは、それぞれ独立に、A、C、GまたはTもしくはUであり、X1およびX2は、それぞれ0〜50個のヌクレオチドを表し、かつ、X1およびX2は相補的に結合して0〜50塩基対のステムを形成する)
モチーフMkを有するアプタマー:
5’−X3−(N)m−Mk−(N)n−X4−3’(ここで、Mkは、RRRDDRNDRGRKW(配列番号208)であり、Rは、それぞれ独立に、AまたはGであり、Dは、それぞれ独立に、A、GまたはTもしくはUであり、Nは、A、C、GまたはTもしくはUであり、Kは、GまたはTもしくはUであり、Wは、AまたはTもしくはUであり、mは、9から15までのいずれかの整数であり、nは、0から4までのいずれかの整数であり、X3およびX4は、それぞれ0〜50個のヌクレオチドを表し、かつ、X3およびX4は相補的に結合して0〜50塩基対のステムを形成する)
実施例3および4の結果から予測されたモチーフのうち、マイクロベシクル結合能に重要であるループ構造部分についてさらに詳細に解析するために、ステム構造部分を短くした以下のアプタマーを合成した。5’末端にはビオチン標識を導入した。
Sモチーフアプタマー: 5’−ggccACGACGCGGAGGUGUGGGGGAUCGUggcc−3’(配列番号209)
Kモチーフアプタマー: 5’−GGUAGAUACGAUGGAAGAGGGAAAGGGAGGGUUCUACC−3’(配列番号210)
(ここで、小文字はDNAを、大文字はRNAを表す。また、UとCはフッ素により修飾されている。また、下線は、モチーフMs(配列番号207)またはモチーフMk(配列番号208)に相当する部分を示す。)
Sモチーフアプタマーと、市販の抗マイクロベシクル抗体のマイクロベシクルに対する結合能を比較するために、ドットブロットを行った。抗マイクロベシクル抗体として、抗CD63抗体(MX−49.129.5、サンタクルーズバイオテクノロジー社製)を用いた。CD63はエキソソームのマーカータンパク質として知られており、抗CD63抗体は、エキソソームの検出・単離のために一般的に使用されている。
Sモチーフアプタマー(配列番号209)とKモチーフアプタマー(配列番号210)の、マイクロベシクルに対する結合能を、表面プラズモン共鳴法により評価した。具体的には、表面プラズモン共鳴測定装置BIACORE X(GEヘルスケア・ジャパン社製)とストレプトアビジンでコートされたセンサーチップ(センサーチップSA、GEヘルスケア・ジャパン社製)を使用した。約150RUのSモチーフアプタマーまたはKモチーフアプタマーをチップに結合させた。アナライトとなる293T細胞由来またはHeLa S3細胞由来のマイクロベシクルは、実施例1と同様の手順により調製したものについて、約6mg/mlを1として、1/40、1/80、1/120、1/140、および1/160の濃度に調製し、結合レベルの測定に使用した。ランニングバッファーには、0.005%Tween20/結合バッファーを用いた(流速10μl/ml)。
Sモチーフアプタマー(配列番号209)とKモチーフアプタマー(配列番号210)について、円偏光二色性(CD)スペクトルを測定することにより、モチーフの立体構造を解析した。具体的には、J−820(日本分光社製)により、0.1cm光路長の石英セルを用いて、25℃、220〜320nmにて測定した。バッファーは、20mMトリス塩酸塩(pH7.5)、150mM塩化ナトリウム、0.5mM塩化マグネシウム、1.5mM塩化カルシウムをベースとし、塩化カリウムを終濃度0.0、0.1、0.3、1.0、5.0、10.0、50.0および100mMになるように加えた。また、バッファーを20mMトリス塩酸塩(pH7.5)とし、塩化カリウムを終濃度0.0、0.1、0.3、1.0、5.0、10.0、50.0および100mMになるように加えた場合も評価した。
次いで、Sモチーフアプタマー(配列番号209)とKモチーフアプタマー(配列番号210)のマイクロベシクルに対する結合能が、カリウムイオン濃度依存的に変化するかどうかを、表面プラズモン共鳴法により解析した。実施例1と同様の手順により調製した293T細胞由来のマイクロベシクル(81mg/ml)と、10mM塩化カリウムを含むまたは含まない結合バッファーとを用い、実施例7と同様の条件にて測定を行った。
Claims (10)
- 下記の塩基配列:
5’−X1−CGCGGAGGUGUGGGGGA−X2−3’
(ここで、X1およびX2は、それぞれ5〜25個のヌクレオチドを表し、かつ、X1およびX2は相補的に結合して5〜25塩基対のステムを形成する)
を含む、マイクロベシクルに対して結合能を有する核酸アプタマー。 - 下記の塩基配列:
5’−X3−UACGAUGGAAGAGGGAAAGGGAGGGU−X4−3’
(ここで、X3およびX4は、それぞれ5〜25個のヌクレオチドを表し、かつ、X3およびX4は相補的に結合して5〜25塩基対のステムを形成する)
を含む、マイクロベシクルに対して結合能を有する核酸アプタマー。 - 前記ステムがミスマッチまたはバルジを含む、請求項1または2に記載の核酸アプタマー。
- 3’および/または5’末端が修飾されている、請求項1〜3のいずれか1項に記載の核酸アプタマー。
- 前記マイクロベシクルの直径が10〜200nmである請求項1〜4のいずれか1項に記載の核酸アプタマー。
- 前記マイクロベシクルがエキソソームである請求項5に記載の核酸アプタマー。
- 請求項1〜6のいずれか1項に記載の核酸アプタマーを用いて、マイクロベシクルを検出する方法。
- 請求項1〜6のいずれか1項に記載の核酸アプタマーを用いて、マイクロベシクルを単離する方法。
- 請求項1〜6のいずれか1項に記載の核酸アプタマーと、薬物とを含む、薬物送達システム。
- マイクロベシクルをさらに含む、請求項9に記載の薬物送達システム。
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