JP6898236B2 - 核酸合成のための方法および装置 - Google Patents
核酸合成のための方法および装置 Download PDFInfo
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- JP6898236B2 JP6898236B2 JP2017529593A JP2017529593A JP6898236B2 JP 6898236 B2 JP6898236 B2 JP 6898236B2 JP 2017529593 A JP2017529593 A JP 2017529593A JP 2017529593 A JP2017529593 A JP 2017529593A JP 6898236 B2 JP6898236 B2 JP 6898236B2
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Description
本発明は、例えば、以下の項目を提供する。
(項目1)
オリゴヌクレオチドを合成する方法であって、前記方法は、
ヌクレオチジルトランスフェラーゼ酵素の存在下、核酸鋳型の非存在下で、固体支持体に付着しているオリゴヌクレオチドをヌクレオチド類似体に曝露し、その結果前記ヌクレオチド類似体が前記オリゴヌクレオチドに組み込まれるステップを含み、
前記ヌクレオチド類似体は、阻害剤が切断可能なリンカーの切断によって除去されるまで前記阻害剤へ前記切断可能なリンカーによって結合されるヌクレオチドを含み、前記阻害剤は前記ヌクレオチジルトランスフェラーゼが前記オリゴヌクレオチドへのヌクレオチドまたはさらなるヌクレオチド類似体の組み込みを触媒することを立体配置的に防止する、方法。
(項目2)
前記ヌクレオチド類似体が、以下の構造
(項目3)
前記ヌクレオチド類似体が、以下の構造
(項目4)
前記阻害剤が、以下の構造
を含む、項目1に記載の方法。
(項目5)
前記阻害剤が、ポリペプトイドを含む、項目1に記載の方法。
(項目6)
前記阻害剤が、ポリマーを含む、項目1に記載の方法。
(項目7)
前記ポリマーが、ポリエチレングリコールを含む、項目6に記載の方法。
(項目8)
前記阻害剤が、タンパク質である、項目1に記載の方法。
(項目9)
前記阻害剤が、ナノ粒子である、項目1に記載の方法。
(項目10)
前記阻害剤が、直径50Å超の巨大分子である、項目1に記載の方法。
(項目11)
前記固体支持体に付着している前記核酸が、pH約6.5〜8.5を有する水溶液の存在下で前記ヌクレオチド類似体に曝露される、項目1に記載の方法。
(項目12)
前記固体支持体に付着している前記核酸が、約35〜39℃の温度で水溶液の存在下で前記ヌクレオチド類似体に曝露される、項目1に記載の方法。
(項目13)
前記固体支持体が、ビーズ、ウェルまたはPEGである、項目1に記載の方法。
(項目14)
前記核酸が、一本鎖である、項目1に記載の方法。
(項目15)
前記切断可能なリンカーが、前記ヌクレオチド類似体から切断されるときに環状の副産物を形成する部分を含む、項目1に記載の方法。
(項目16)
天然のヌクレオチドを産生するために前記切断可能なリンカーを切断するステップと;
ヌクレオチジルトランスフェラーゼ酵素の存在下、核酸鋳型の非存在下で、前記天然のヌクレオチドを第2のヌクレオチド類似体に曝露するステップと
をさらに含む、項目1に記載の方法。
(項目17)
前記オリゴヌクレオチドをエキソヌクレアーゼ酵素に曝露するステップをさらに含む、項目1に記載の方法。
(項目18)
前記ヌクレオチド類似体および前記ヌクレオチジルトランスフェラーゼ酵素を含む水溶液を提供するステップをさらに含む、項目1に記載の方法。
(項目19)
前記阻害剤が、荷電部分を含む、項目1に記載の方法。
(項目20)
前記ヌクレオチド類似体が、リボース糖またはデオキシリボース糖を含む、項目1に記載の方法。
(項目21)
ヌクレオチド基質が、アデニン、グアニン、シトシン、チミンおよびウラシルからなる群から選択される塩基を含む、項目1に記載の方法。
(項目22)
前記ヌクレオチジルトランスフェラーゼが、配列番号1、配列番号3、または配列番号5と少なくとも約90%同一のタンパク質配列を含む、項目1に記載の方法。
(項目23)
前記ヌクレオチジルトランスフェラーゼが、配列番号2、配列番号4、または配列番号6と少なくとも約90%同一のヌクレオチド配列を有する生物に由来する、項目1に記載の方法。
(項目24)
前記ヌクレオチド類似体が、液滴で送達される、項目1に記載の方法。
NTP−リンカー−阻害剤
の形態のものであり、そこで、NTPはヌクレオチド三リン酸(すなわち、dNTPまたはrNTP)であり、リンカーは塩基のピリジンまたはピリミジン間の切断可能なリンカーであり、阻害剤は、酵素が次のヌクレオチドを組み込むことを防止する基である。各ステップにおいて、新しいヌクレオチド類似体が、成長中のポリヌクレオチド鎖に組み込まれ、阻害基(inhibitor group)によって、酵素がさらなるヌクレオチドを付加することが遮断される。酵素が止まったら、過剰なヌクレオチド類似体を成長中の鎖から除去することができ、阻害剤をNTPから切断することができ、鎖に次のヌクレオチドを付加するために新しいヌクレオチド類似体を導入することができる。このステップを連続して繰り返すことにより、所望の長さおよび配列のヌクレオチド配列をすばやく構築することができる。ポリヌクレオチド合成にヌクレオチジルトランスフェラーゼを使用する長所には:1)鋳型非依存的重合において一本鎖(ss)開始プライマーを使用する3’伸長活性、2)高効率の様式でプライマーを伸長させて数千のヌクレオチドを付加する能力、および3)効率的な基質としての多種多様な修飾および置換NTPの受容、が挙げられる。加えて、本発明は、ヌクレオチジルトランスフェラーゼの基質であるイニシエーター配列を使用することができる。イニシエーターは、固体支持体に付着され、酵素の結合部位として用いられる。イニシエーターは、好ましくは酵素に対する汎用のイニシエーター、例えばホモポリマー配列であり、固体支持体上で再利用可能であり、形成されたオリゴヌクレオチドがイニシエーターから切断可能である。
類似体
酵素
本発明に特に重要(import)なことは、ポリメラーゼ活性を有するトランスフェラーゼ、例えばターミナルデオキシヌクレオチジルトランスフェラーゼ(TdT)は、ヌクレオチド鎖の3’末端へのデオキシリボヌクレオチドの付加を触媒する能力があり、それによってDNAヌクレオチドの鎖長が増加する。TdTは、DNA鎖の成長末端への1〜2つのリボヌクレオチドの付加しか触媒しないことになり、これは部位特異的DNA−RNAキメラポリヌクレオチドの構築に有用になり得る。特に、操作した大腸菌から供給される仔ウシ胸腺TdTは、本発明での使用に適しており、市販品、例えばThermo Scientific(ピッツバーグ、PA)から入手可能である。仔ウシTdTに対応するアミノ酸配列を、配列番号1として表1に挙げる。
固体状態合成
合成機
参照による援用
等価物
Claims (17)
- オリゴヌクレオチドを合成する方法であって、前記方法は、
ヌクレオチジルトランスフェラーゼ酵素の存在下、核酸鋳型の非存在下で、固体支持体に付着しているオリゴヌクレオチドをヌクレオチド類似体に曝露し、その結果前記ヌクレオチド類似体が前記オリゴヌクレオチドに組み込まれるステップを含み、
前記ヌクレオチド類似体は、阻害剤が切断可能なリンカーの切断によって除去されるまで前記阻害剤へ前記切断可能なリンカーによって結合されるヌクレオチドを含み、前記阻害剤は前記ヌクレオチジルトランスフェラーゼによる前記オリゴヌクレオチドへの天然のヌクレオチドまたはヌクレオチド類似体のいずれの組み込みについても触媒することを立体配置的に防止し、
前記阻害剤が、直径50Å超のタンパク質である、方法。 - 前記固体支持体に付着している前記核酸が、pH6.5〜8.5を有する水溶液の存在下で前記ヌクレオチド類似体に曝露される、請求項1に記載の方法。
- 前記固体支持体に付着している前記核酸が、35〜39℃の温度で水溶液の存在下で前記ヌクレオチド類似体に曝露される、請求項1に記載の方法。
- 前記固体支持体が、ビーズ、ウェルまたはPEGである、請求項1に記載の方法。
- 前記核酸が、一本鎖である、請求項1に記載の方法。
- 前記切断可能なリンカーが、前記ヌクレオチド類似体から切断されるときに環状の副産物を形成する部分を含む、請求項1に記載の方法。
- 天然のヌクレオチドを産生するために前記切断可能なリンカーを切断するステップと;
ヌクレオチジルトランスフェラーゼ酵素の存在下、核酸鋳型の非存在下で、前記天然のヌクレオチドを第2のヌクレオチド類似体に曝露するステップと
をさらに含む、請求項1に記載の方法。 - 前記オリゴヌクレオチドをエキソヌクレアーゼ酵素に曝露するステップをさらに含む、請求項1に記載の方法。
- 前記ヌクレオチド類似体および前記ヌクレオチジルトランスフェラーゼ酵素を含む水溶液を提供するステップをさらに含む、請求項1に記載の方法。
- 前記阻害剤が、荷電部分を含む、請求項1に記載の方法。
- 前記ヌクレオチド類似体が、リボース糖またはデオキシリボース糖を含む、請求項1に記載の方法。
- ヌクレオチド基質が、アデニン、グアニン、シトシン、チミンおよびウラシルからなる群から選択される塩基を含む、請求項1に記載の方法。
- 前記ヌクレオチジルトランスフェラーゼが、配列番号1、配列番号3、または配列番号5と少なくとも90%同一のタンパク質配列を含む、請求項1に記載の方法。
- 前記ヌクレオチジルトランスフェラーゼが、配列番号2、配列番号4、または配列番号6と少なくとも90%同一のヌクレオチド配列を有する生物に由来する、請求項1に記載の方法。
- 前記ヌクレオチド類似体が、液滴で送達される、請求項1に記載の方法。
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