JP6880034B2 - 特異的rnaの安定化のための汎用法 - Google Patents
特異的rnaの安定化のための汎用法 Download PDFInfo
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- JP6880034B2 JP6880034B2 JP2018533890A JP2018533890A JP6880034B2 JP 6880034 B2 JP6880034 B2 JP 6880034B2 JP 2018533890 A JP2018533890 A JP 2018533890A JP 2018533890 A JP2018533890 A JP 2018533890A JP 6880034 B2 JP6880034 B2 JP 6880034B2
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Description
「増幅試薬」は、核酸の増幅を可能にする化学的又は生化学的成分である。そのような試薬には、核酸ポリメラーゼ、緩衝液、モノヌクレオチド、例えばヌクレオシド三リン酸、オリゴヌクレオチドが、例えばオリゴヌクレオチドプライマー、塩及びそれらの各溶液、検出プローブ、色素などとして含まれるが、これらに限定されるものではない。
・55℃〜90℃、又は65℃〜85℃、又は70℃〜80℃、又は約75℃の融解温度
・最高500塩基もしくは塩基対、又は50〜300塩基もしくは塩基対、又は100〜200塩基もしくは塩基対、又は約180塩基もしくは塩基対の長さ
・30%〜70%、又は40%〜60%、又は約50%のGC含有量。
a)CS5DNAポリメラーゼ
b)CS6DNAポリメラーゼ
c)Thermotoga maritimaのDNAポリメラーゼ
d)Thermus aquaticusのDNAポリメラーゼ
e)Thermus thermophilusのDNAポリメラーゼ
f)Thermus flavusのDNAポリメラーゼ
g)Thermus filiformisのDNAポリメラーゼ
h)Thermus sp.sps17のDNAポリメラーゼ
i)Thermus sp.Z05のDNAポリメラーゼ
j)Thermotoga neapolitanaのDNAポリメラーゼ
k)Termosipho africanusのDNAポリメラーゼ
l)Thermus caldophilusのDNAポリメラーゼ。
・HIV:最高60cp/ml、最高50cp/ml、最高40cp/ml、最高30cp/ml、最高20cp/ml、又は最高15cp/ml
・HBV:最高10IU/ml、最高7.5IU/ml、又は最高5IU/ml
・HCV:最高10IU/ml、最高7.5IU/ml、又は最高5IU/ml
・WNV I:最高20cp/ml、最高15cp/ml、又は最高10cp/ml
・WNV II:最高20cp/ml、最高15cp/ml、最高10cp/ml、又は最高5cp/ml
・JEV:最高100cp/ml、最高75cp/ml、最高50cp/ml、又は最高30cp/ml
・SLEV:最高100cp/ml、最高75cp/ml、最高50cp/ml、最高25cp/ml、又は最高10cp/ml。
T’=10(a(CTQS−CT標的)2+b(CTQS−CT標的)+c)
コンピュータ設計ツール(silico design tool)を使用してRNA配列に対して相補的オリゴヌクレオチドを設計した。合計10のオリゴヌクレオチドを設計して、長さは14〜26塩基で変動し、Tm計算値は49.9〜57.9Cの範囲であり、関心対象の配列をカバーした。これらの配列を、3’末端でのホスフェート部分でさらに修飾した10個のオリゴヌクレオチドの配列及び融解温度を表1に示す。
RNA転写物及び武装化RNAサンプルを、100mMのKClを含有するTris.HCl(pH7.0)中300コピー/マイクロリットルの濃度で調製した。安定化のための相補的オリゴヌクレオチドプール(COPS)と称する補体プールをサンプルに0、0.1、1、又は10nMの最終濃度で添加した。サンプルを2〜8℃、37℃又は45℃で18日間インキュベートした。
5マイクロリットルの各サンプルをTaqman(登録商標)に基づくRT−PCRによって増幅した。PCR反応混合物を以下の最終濃度で96ウェルプレート上で調製した:60mMのTricine(pH8.3)、120mMの酢酸カリウム、3%グリセロール、5.4%DMSO、0.015%Tween20、各々400μMのdATP、dCTP及びdGTP、800μMのdUTP、各々600nMのプライマー、100nMのプローブ、標的RNA転写物又は武装化RNA(1,500コピー)、900単位/mLのZO5D DNAポリメラーゼ(5`ヌクレアーゼ活性を有する)、200単位/mLのUNG、44μMのEDTA、及び3.3mMの酢酸マンガン。逆転写、増幅及び分析は、Roche LightCycler(登録商標)480機器(Roche Molecular Systems, Pleasanton, CA)を使用して実施した。以下の温度プロフィールを使用した:50℃で2分、94℃で5秒、55℃で2分、60℃で6分、65℃で4分、95℃(10秒)から55℃(15秒)を2サイクルと続いて91℃(5秒)から65℃(15秒)のサイクルを45回。これらの実験結果を図2及び図3に示す。容易にわかるように、COPSオリゴヌクレオチドを有しないサンプルと比較して以前のCtによって示されるように、相補的オリゴヌクレオチドプールの存在下で、RNA転写物及び武装化RNAはどちらもより安定である。
1つの反応では、配列番号1、3、5、7及び10に対応するCOPSのみを添加し(セットA)、そして別の反応では、配列番号2、4、6、8及び9に対応するCOPSのみを添加する(セットB)以外は、実施例2においてと同様にして、RNA転写物及び武装化RNAサンプルを調製する。計算により、セットAがRNA転写物/武装化RNA配列の52%をカバーし、一方、セットBはRNA転写物/ 武装化RNA配列の48%をカバーすることが示される。45℃で18日インキュベーションした後、COPSの不在下又はセットA COPSもしくはセットB COPSの存在下でのRNA安定性は、実施例3で記載するようなRT−PCRにより各反応のCt値を決定することによって比較することができる。
武装化RNAを1500コピー/マイクロリットルで調製した以外は、実施例2においてと同様にRNA転写物及び武装化RNAサンプルを調製した。COPSを次いでサンプルに0、0.1、1又は10nMの最終濃度で添加し、サンプルを4℃、37℃又は45℃で12週間インキュベーションした。RT−PCRによるRNA安定性の判定を実施例3に記載したようにして実施した。37℃及び45℃でのインキュベーションで非武装化及び武装化RNAの両方についてCOPSの存在で安定性は有意に改善された。図4は、非武装化RNAテンプレートのRT−PCR成長曲線の結果を示す。COPSの不在下(一番上のグラフ)で、45℃でインキュベートしたサンプルは、4℃でインキュベートしたサンプルと比較して、Ct値の10周期遅延を示した。対照的に、10nMのCOPSの存在下(一番下のグラフ)で、45℃サンプルは1.4周期遅延しか示さず、このことは、RNA安定性において8.6周期、すなわち約400倍の改善を実証する。武装化RNA実験について、7.4周期、すなわち約200倍の改善が観察された(データは不掲載)。
Accuplex(SeraCare Life Sciences, Milford MA)は、タンパク質コート及び脂質二重層の両方を含む複製欠損哺乳動物ウイルス様粒子の内部に関心対象のRNA分子を封入することができる組換え技術である。Accuplex粒子内部のRNAの安定化についてCOPSの有用性を試験するために、実施例1で記載する相補的オリゴヌクレオチドを設計し調製するために使用したRNA対照配列pEF070をAccuplexカプセル化一本鎖RNAのカスタム調製のためのSeraCareに提供した。COPSを次いでAccuplex−RNAサンプルに10nM濃度で添加し、そしてサンプルを4℃、37℃又は45℃で71日間インキュベートした。RT−PCRによるRNA安定性の判定を実施例3に記載するようにして実施し、試験結果を図6に示す。71日のインキュベーション後、COPSの不在と存在との間のCp値Δは37℃について4.3(30.9〜26.6)そして45℃について8.3(35.3〜27.0)であり、高温で保存されるAccuplex粒子におけるRNAの分解の低減に際してのCOPSの有効性を明らかに示す。
HIV−1GAG、HIV−1LTR、及びHIV−2LTR領域のセグメントに対応する3つのRNA配列を、それらの対応するCOPSの安定化効果を試験するためのRNAテンプレートとして使用した。関心対象の核配列をカバーするために、HIV−GAGについて9、HIV−1LTRについて8、そしてHIV−2LTRについて8の合計25のオリゴヌクレオチドを設計した。25のオリゴヌクレオチドは、17〜26塩基で長さが様々であり、それらの配列を表2に示す。
Claims (12)
- 増幅反応で増幅される一本鎖RNAテンプレートのセグメントの分解を防止又は低減する方法であって、
a)前記一本鎖RNAテンプレートを提供するステップと、
b)前記一本鎖RNAテンプレートの前記セグメントを、増幅される前記一本鎖RNAテンプレートの前記セグメントと完全に又は部分的に相補的である配列を有する1以上のオリゴヌクレオチドとハイブリダイズさせるステップと、
c)反応条件下で前記一本鎖RNAテンプレートの前記セグメントを逆転写し増幅するステップであって、前記反応条件によって、前記1以上のオリゴヌクレオチドは逆転写及び増幅を妨害せず、そして前記反応条件によって、前記1以上のオリゴヌクレオチドからの各々のオリゴヌクレオチドは、11ヌクレオチド〜50ヌクレオチドの長さであり、そして増幅中に使用される伸長温度よりも少なくとも5℃低い融解温度を有することによって特徴付けられ、そしてここで前記1以上のオリゴヌクレオチドからの各々のオリゴヌクレオチドの配列は前記1以上のオリゴヌクレオチドからの他のオリゴヌクレオチドの配列とは重複せず、そして前記1以上のオリゴヌクレオチドが逆転写及び増幅中に使用されるプライマー及びプローブの濃度よりも少なくとも50倍低い濃度で存在することによって特徴づけられる、ステップと
を含む、前記方法。 - 前記1以上のオリゴヌクレオチドが、増幅される前記一本鎖RNAテンプレートの前記セグメントの48%超とハイブリダイズするか、又は
前記1以上のオリゴヌクレオチドが、増幅される前記一本鎖RNAテンプレートの前記セグメント全体とハイブリダイズする、請求項1に記載の方法。 - 前記一本鎖RNAテンプレートがケージ化されている、請求項1又は2に記載の方法。
- 前記一本鎖RNAテンプレートが、カプセル化、キャプシド形成、トラッピング、及び細胞の内部に存在すること、からなる群から選択される手段によってケージ化されている、請求項3に記載の方法。
- ステップb)とステップc)との間に、前記一本鎖RNAテンプレートを単離又は精製するステップをさらに含む、請求項1〜4のいずれか1項に記載の方法。
- 増幅反応の間に核酸サンプル中の試験されるRNA配列の存在を検出する方法であって、
a)前記核酸サンプルを提供するステップと、
b)試験されるRNA配列の検出及び/又は定量化において標準としての役割を果たす核酸標準を提供するステップであって、前記核酸標準が、一本鎖RNA対照配列と、その配列が前記一本鎖RNA対照配列のセグメントに対して完全又は部分的に相補的である1以上のオリゴヌクレオチドとを含む、ステップと、
c)前記核酸サンプルと前記核酸標準とを混合するステップと、
d)前記試験されるRNA配列と前記一本鎖RNA対照配列の前記セグメントの両方の逆転写及び増幅を実施するための条件を提供するステップであって、前記条件下で、前記1以上のオリゴヌクレオチドは逆転写及び増幅を妨害せず、それによって、前記1以上のオリゴヌクレオチドからの各々のオリゴヌクレオチドが、11ヌクレオチド〜50ヌクレオチドの長さであり、そして増幅の間で使用される伸長温度よりも少なくとも5℃低い融解温度を有することによって特徴付けられ、そしてここで前記1以上のオリゴヌクレオチドからの各々のオリゴヌクレオチドの配列は前記1以上のオリゴヌクレオチドからの他のオリゴヌクレオチドの配列とは重複せず、そして前記1以上のオリゴヌクレオチドが逆転写及び増幅の間で用いられるプライマー及びプローブの濃度よりも少なくとも50倍低い濃度で存在することによって特徴づけられるステップと、
e)前記試験されるRNA配列からの増幅産物と、前記一本鎖RNA対照配列の前記セグメントからの増幅産物を検出するステップと
を含む、前記方法。 - 前記1以上のオリゴヌクレオチドが前記一本鎖RNA対照配列の前記セグメントの48%超とハイブリダイズするか、又は
前記1以上のオリゴヌクレオチドが前記一本鎖RNA対照配列の前記セグメント全体とハイブリダイズする、請求項6に記載の方法。 - 前記一本鎖RNA対照配列がケージ化されている、請求項6〜7のいずれか1項に記載の方法。
- ステップc)とステップd)との間で、前記一本鎖RNA対照配列を単離又は精製するステップをさらに含む、請求項6〜8のいずれか1項に記載の方法。
- 前記1以上のオリゴヌクレオチドが、その配列が配列番号1〜10、11〜19、20〜27、及び28〜35からなる群から選択されるオリゴヌクレオチドの群を含む、請求項1〜9のいずれか1項に記載の方法。
- 増幅反応で増幅される試験されるRNA配列の検出及び/又は定量化において標準としての役割を果たす核酸標準であって、
一本鎖RNA対照配列と、
その配列が、前記一本鎖RNA対照配列に対して完全又は部分的に相補的であり、前記一本鎖RNA対照配列全体にハイブリダイズする1以上のオリゴヌクレオチドと
を含み、
前記1以上のオリゴヌクレオチドからの各オリゴヌクレオチドは、前記増幅反応の間に用いられる伸長温度よりも少なくとも5℃低い融解温度を有することによって特徴付けられ、かつ前記1以上のオリゴヌクレオチドからの各オリゴヌクレオチドは、増幅反応の間で使用されるプライマー及びプローブの濃度よりも少なくとも50倍低い濃度で存在することによって特徴付けられ、ここで前記1以上のオリゴヌクレオチドからの各オリゴヌクレオチドが11ヌクレオチド〜50ヌクレオチドの長さである、核酸標準。 - 前記1以上のオリゴヌクレオチドが、その配列が配列番号1〜10、11〜19、20〜27、及び28〜35からなる群から選択されるオリゴヌクレオチドの群を含む、請求項11に記載の核酸標準。
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