JP4567673B2 - ポリメラーゼ阻害剤およびその使用方法 - Google Patents
ポリメラーゼ阻害剤およびその使用方法 Download PDFInfo
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- JP4567673B2 JP4567673B2 JP2006509580A JP2006509580A JP4567673B2 JP 4567673 B2 JP4567673 B2 JP 4567673B2 JP 2006509580 A JP2006509580 A JP 2006509580A JP 2006509580 A JP2006509580 A JP 2006509580A JP 4567673 B2 JP4567673 B2 JP 4567673B2
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- Prior art keywords
- nucleic acid
- polymerase
- polymerase inhibitor
- stranded
- double
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- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Description
本出願は、米国特許仮出願第60/459,672号に対して優先権を主張するものであり、その全体が本明細書中に参考として援用される。
本発明は、核酸ポリメラーゼ活性を阻害する方法および試薬に関するものである。より具体的には、本発明は、低温において核酸ポリメラーゼによる非特異的核酸の伸長または増幅を防止する方法および核酸に関するものである。
ポリメラーゼ連鎖反応(PCR)は、核酸を増幅するための多用途かつ強力な技術であることが分かっている。PCRは、天然のDNAポリメラーゼまたは組換えDNAポリメラーゼが標的核酸を高レベルに再生成する能力を利用する。理論的には、この手順は、単一コピーのDNAの対数再生成(増幅)を生成する能力を有する。しかしながら、増幅工程中の多数の要因によってPCRプロセスの感度に障害が生じ、結果的に顕著な感度の低下が生じる。主要な問題の1つが、一般に「プライマーダイマー」として知られている、反応中の非特異的産物の発生である。これらの産物が形成すると、これらの産物はプライマーおよびデオキシリボヌクレオシド三リン酸(dNTPs)の両方を反応液から除去してしまい、これにより、所望の標的の増幅レベルが低下し、同時に反応の感度も低下する。
本発明は、核酸ベースのポリメラーゼ阻害剤、および核酸増幅反応における該阻害剤の使用方法を提供する。一実施形態は、核酸配列を含むポリメラーゼ阻害剤を提供し、少なくともその一部は、この核酸配列の融点またはそれ未満において二本鎖である。いくつかの実施形態において、この核酸配列の二本鎖部分は、この核酸配列がポリメラーゼによって実質的に伸長され得ないことを除いて、伸長のためのテンプレートとしてポリメラーゼによって認識されるに十分な長さである。さらに、いくつかの実施形態において、上記核酸が二本鎖である場合に、この核酸の3’末端の核酸の少なくとも1つが、この核酸の5’末端の核酸の少なくとも1つと対をなす必要はない。
図1Aおよび1Bはヘアピンデコイである。ヘアピンデコイは、自己相補配列を含む単一のオリゴヌクレオチドであり、この自己相補配列は、自己ハイブリダイズして、5’伸長(すなわち尾部)およびポリメラーゼによって伸長不可能な3’末端と部分的に二本鎖を形成する。
本発明は、核酸ベースのポリメラーゼ阻害剤を提供する。このポリメラーゼ阻害剤は、所定の温度未満、一般的には非特異的プライミングおよび伸長が生じる温度未満ではポリメラーゼ活性を阻害するが、所定の温度を超えると、一般的にはプライマーが標的核酸に特異的にアニーリングする温度を超えると、ポリメラーゼ活性を可能にする。本明細書中で使用される場合、「核酸ベースのポリメラーゼ阻害剤」、またはいくつかの例においては単純に「ポリメラーゼ阻害剤」は、少なくとも部分的に核酸から構成されるポリメラーゼ阻害剤を意味する。いくつかの実施形態において、特に反応混合物の温度が所定の温度未満である場合は、上記ポリメラーゼ阻害剤の核酸部分は、少なくとも部分的に二本鎖である。上記核酸の部分的に二本鎖である部分は、ポリメラーゼによって、少なくとも潜在的に伸長の基質であると認識されるに十分な長さを有している。核酸ベースのポリメラーゼ阻害剤は、全体またはほぼ全体が核酸から構成され得る。一方、他の実施形態において、核酸は、非核酸部分(例えば、タンパク質、保護基、標識など)に連結され得る。核酸の二本鎖部分は、もし存在するならば、例えば、ヘアピン様構造もしくはステムループ構造、または、互いにアニーリングした2つ以上の別々のオリゴヌクレオチドを形成することによって、自身とアニーリングした単一のオリゴヌクレオチドから生じ得る。一般に、ポリメラーゼ阻害剤は、2つのオリゴヌクレオチドを含む。なぜなら、その溶融する挙動は、より好適であり、自己アニーリングする単一のオリゴヌクレオチドよりも狭い範囲を有する、より迅速な変性速度を可能にするからである。従って、核酸ベースのポリメラーゼ阻害剤は、2つ以上の個別の実体から構成され得る。この2つ以上の個別の実体は、両者とも核酸であってもよく、一方が、リガンド(例えば、タンパク質または核酸模倣物)であってもよく、このリガンドは核酸ベースのポリメラーゼ阻害剤の他の部分と結合する。核酸の二本鎖部分を形成する別々の2つのオリゴヌクレオチドは、同じ配列または異なる配列を有し得る。自身でまたは他のオリゴヌクレオチドとアニーリングするオリゴヌクレオチドを提供することは、当該分野において公知の種々の手段(互いに相補的な核酸部分を提供すること、または核酸内に回文配列を提供することを含む。)を介して達成され得る。これらの実施形態のいくつかにおいて、核酸ベースのポリメラーゼ阻害剤は、伸長された5’尾部および遊離3’末端を有することができる。好ましい長さの範囲は5’尾部が7〜16bpであり、5〜16bpの二重鎖領域を、10、または最もよく機能すると思われる10より大きい長さで試験した。
ここで、Aは、糖またはポリマー骨格の他の部位への結合点であり、Rは、H、または置換アルキル基もしくは非置換アルキル基である。水素結合を利用する他の非標準塩基が調製され得ること、および塩基の非水素結合原子において官能基を取り込むことによって、上記で特定した非標準塩基を修飾できることは、理解されるであろう。図3から9に示されているこれらの非標準塩基を示すために、以下の記号が使用される:Xはiso−Cを示し、Yはiso−Gを示す。
〔実施例1:リアルタイムPCRにおけるプライマーダイマー形成に対するデコイの影響の例証〕
反応物を以下のように準備した:1ulの10×PCR緩衝液(100mMのBTP(pH9.1)、400mMのKAc、20mMのMgCl2、1mg/mlのBSA)、各100uMのdATP、dTTP、dCTP、dGTP(Promega社)、3uMのdabcyl iGTP(EraGen Biosciences社)、200nMの順方向PCRプライマー CL025(5’−FAM−TXGATAGCAACAATTCATCTACAGA)、200nMの逆方向PCRプライマー CL026(5’ATGGGTAGTGAATGATCTTGTTTC)、1UのKlenTaq(Ab peptides社)、および5ulの合成DNA標的 CL021(5’TCAGATAGCAACAATTCATCTACAGACCCAATTAGCAGTGGAGAAACAAGATCATTCACTACCCATTTCTTAACTTATCCCAAGATAGGACTTCTGTACA)。段階希釈の濃度は、100,000〜0.1分子の範囲である。デコイの非存在下(図5A)、5uMのMM309/310(図5B)、5uMのMM309/311(図5C)、および5uMのMM309/312(図5D)の存在下においては、標的コントロールはいずれも増幅されなかった。PCR熱サイクリングを、iCycler(Bio−Rad社)において、次のサイクリング条件を用いて行った:94°変性で2分間、PCR60回:光学測定を用いて、94°で1秒間、58°で1秒間;72°で20秒。PCRサイクリングの後、融解解析を行った。サンプルを60°〜95°の間で0.5°上昇させる毎に光学測定を用いて加熱した。
Claims (17)
- 核酸を含むポリメラーゼ阻害剤であって、該核酸の一部は、該核酸の融点またはそれ未満において二本鎖であり、該核酸が該ポリメラーゼによって伸長され得ないことを除いて、該核酸の該二本鎖部分が、伸長のためのテンプレートとしてポリメラーゼによって認識されるに十分な長さであり、かつ、該核酸の少なくとも5’末端のヌクレオチドが、該核酸の融点またはそれ未満において一本鎖である、ポリメラーゼ阻害剤。
- 特定のポリメラーゼに特異的でない、請求項1に記載のポリメラーゼ阻害剤。
- 請求項1に記載のポリメラーゼ阻害剤であって、該ポリメラーゼ阻害剤の阻害活性が該ポリメラーゼ阻害剤の核酸配列に依存しない、ポリメラーゼ阻害剤。
- 請求項1に記載のポリメラーゼ阻害剤であって、該ポリメラーゼ阻害剤の核酸が、目的の標的核酸に対するプライマーとして作用し得ない、ポリメラーゼ阻害剤。
- 請求項1に記載のポリメラーゼ阻害剤であって、該ポリメラーゼ阻害剤の核酸が、目的の標的核酸を含む核酸の一部を形成しない、ポリメラーゼ阻害剤。
- 核酸から構成される、請求項1に記載のポリメラーゼ阻害剤。
- 上記核酸の3’末端のヌクレオチドが、上記ポリメラーゼによる該3’末端のヌクレオチドの伸長を防止するブロック部分を含む、請求項1に記載のポリメラーゼ阻害剤。
- 上記核酸の融点またはそれ未満において二本鎖である核酸の一部が、それ自身にアニーリングし得る単一の核酸によって形成されている、請求項1に記載のポリメラーゼ阻害剤。
- 上記核酸の融点またはそれ未満において二本鎖である核酸の一部が、部分的に互いにアニーリングする2つの別々の核酸によって形成される、請求項1に記載のポリメラーゼ阻害剤。
- 上記核酸の少なくとも3’末端のヌクレオチドが、該核酸の融点またはそれ未満において一本鎖である、請求項1に記載のポリメラーゼ阻害剤。
- 請求項1に記載のポリメラーゼ阻害剤であって、該ポリメラーゼ阻害剤の核酸部分がDNAまたはDNA模倣物を含む、ポリメラーゼ阻害剤。
- 上記核酸がRNAまたはRNA模倣物を含む、請求項1に記載のポリメラーゼ阻害剤。
- 請求項1に記載のポリメラーゼ阻害剤であって、該ポリメラーゼ阻害剤の核酸部分がエクソヌクレアーゼ分解に耐性である、ポリメラーゼ阻害剤。
- 請求項1に記載のポリメラーゼ阻害剤であって、該ポリメラーゼ阻害剤の核酸部分の二本鎖部分の融点が25℃〜80℃の範囲にある、ポリメラーゼ阻害剤。
- 請求項1に記載のポリメラーゼ阻害剤であって、該ポリメラーゼ阻害剤の核酸部分の二本鎖部分が少なくとも10塩基長である、ポリメラーゼ阻害剤。
- 請求項1〜15のいずれか一項に記載のポリメラーゼ阻害剤の存在下にて核酸増幅反応を行う工程を含む、核酸ポリメラーゼを阻害する方法。
- 請求項1〜15のいずれか一項に記載のポリメラーゼ阻害剤および核酸増幅反応を行なうための1つ以上の追加試薬を含む、核酸ポリメラーゼを阻害するためのキット。
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EP1615942B1 (en) | 2016-03-16 |
WO2004090153A3 (en) | 2005-06-23 |
CA2521127A1 (en) | 2004-10-21 |
US8313932B2 (en) | 2012-11-20 |
CA2521127C (en) | 2014-05-27 |
AU2004227353B2 (en) | 2010-02-25 |
EP1615942A2 (en) | 2006-01-18 |
WO2004090153A2 (en) | 2004-10-21 |
AU2004227353A1 (en) | 2004-10-21 |
US7820808B2 (en) | 2010-10-26 |
EP1615942A4 (en) | 2009-03-25 |
JP2006521824A (ja) | 2006-09-28 |
US20080108124A1 (en) | 2008-05-08 |
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