JP2020511432A - 区画化ペプチドライブラリを生成及びスクリーニングする方法 - Google Patents
区画化ペプチドライブラリを生成及びスクリーニングする方法 Download PDFInfo
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Abstract
Description
本発明は、環状ペプチドとそれをコードするポリヌクレオチドとの共区画化に関する。特に、本発明は、このような区画化環状ペプチドのライブラリ、及びこのようなライブラリから区画化環状ペプチドをスクリーニング、選択、及びソーティングする方法に関する。
油相に分散した水性の液滴内に個々の試料を区画化することは、化学及び生物学における高スループットアッセイにとって強力な方法となる。この場合、液滴は、試験管に相当する。液滴には、ライブラリ構成要素の特定の実験又はプロファイルを評価及び解読するために必要な全てのものが含まれている。バルクエマルション手法により生成された液滴は、サイズが均一ではなく、定量的な読み取りが必要な実験では複雑な問題が生じる。マイクロ流体デバイスにより、毎秒最大数千(数万)の頻度で、高単分散性の水性液滴を生成することができる。通常、直径は、10乃至200ミクロンで、0.5pL乃至4nLの体積に相当する。液滴の形成に加え、マイクロ流体形式では、液滴の分裂、融合、インキュベーション、分析、ソーティング等、他の多数の単位操作が可能となる。
1)ポリヌクレオチド集団、レポータ基質、及びゲル形成剤を含む水性レポータ溶液を乳化して微小液滴とし、前記微小液滴内でレポータ基質を、検出可能なレポータに転換する産物を各ポリヌクレオチドがコードした状態にすること、
2)ゲル形成剤を微小液滴内で固化させ、ポリヌクレオチドと産物により生成された検出可能なレポータとを含むゲルビーズを生成すること、
3)水性微小液滴を解乳化し、水性検出溶液にビーズを再懸濁すること、
4)前記集団中の1個以上のビーズ内で検出可能なレポータを検出、決定、又は測定すること。
a)環状ポリペプチドをコードするポリヌクレオチドを含む区画を形成するステップと、
b)ポリヌクレオチドからポリペプチドを発現させるステップと、
c)ポリペプチドを環化させるステップと、を備える方法が開示される。
a)環状ポリペプチドをコードするポリヌクレオチドを含む区画を形成するステップと、
b)ポリヌクレオチドからポリペプチドを発現させるステップと、
c)ポリペプチドを環化させるステップと、
d)環状ポリペプチドを活性についてスクリーニングするステップと、
e)所望の活性を示す環状ポリペプチドを選択するステップと、を備える方法が開示される。
a)N末端インテイン断片をコードするポリヌクレオチド、それに続く環状ペプチドをコードする配列、それに続くC末端インテイン断片をコードする配列と、
b)環状ポリペプチドと、を備える区画が開示される。
a)マイクロ流体デバイスと、
b)N末端インテイン断片をコードするポリヌクレオチドと、
c)C末端インテイン断片をコードするポリヌクレオチドと、を備えるキットが提供される。
HHHHHHGENLYFKLQAMGMIKIATRKYLGKQNVYDIGVERYHNFALKNGFIASNX〜〜〜〜〜CLSYDTEILTVEYGILPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGCLIRATKDHKFMTVDGQMMPIDEIFERELDLMRVDNLPNGTAANDENYALAA
ここで、X〜〜〜〜〜は、生成される環状ペプチドであり、Xは、C、S、T又は他の任意のアミノ酸であり、「〜」は、環状ペプチド配列のアミノ酸を示す。上記配列の「X」の後に任意の配列を挿入し得ることは、当業者には明らかであろう。配列は、1つ以上のアミノ酸の長さ、実施形態では少なくとも3つ以上のアミノ酸の長さ、好ましくは少なくとも6つのアミノ酸の長さを有し得る。
1:
HHHHHH
例えば、ニッケル-NTAカラム上での精製を支援するオプションのヘキサヒスチジンタグ。「FLAG-TAG」等の他の精製系が考えられ、この場合、ヘキサヒスチジンは、適切なタグ配列に置き換えられる。
2:
GENLYFKLQAMGMIKIATRKYLGKQNVYDIGVERYHNFALKNGFIASN
C末端インテイン断片を含む。
3:
X〜〜〜〜〜
環状ポリペプチド配列。
4:
CLSYDTEILTVEYGILPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGCLIRATKDHKFMTVDGQMMPIDEIFERELDLMRVDNLPNGTAANDENYALAA
N末端インテイン断片を含む。
(1)ポリペプチド自体が、特有の光学特性を有する場合があり、例えば、蛍光性である(例えば、緑色蛍光タンパク質(Lorenz et al., 1991))。
(2)ポリペプチドの光学特性が、リガンドとの結合時に修飾される場合があり、例えば、ポリペプチドの蛍光が、結合時に消失又は増強する((Guixe et al., 1998、Qi and Grabowski, 1998)。
(3)リガンドの光学特性が、ポリペプチドの結合時に変化する場合があり、例えば、リガンドの蛍光が、結合時に消失又は増強する(Voss, 1993、Masui and Kuramitsu, 1998)。
(4)リガンド及びポリペプチドの両方の光学特性が、結合時に修飾される場合があり、例えば、リガンドからポリペプチドへ(又はその逆)の蛍光共鳴エネルギ移動(FRET)があり、励起が「ドナー」吸収波長である場合に「アクセプタ」発光波長での発光が生じる(Heim & Tsien, 1996、Mahajan et al., 1998、Miyawaki et al., 1997)。
(1)例えば、以下のため、基質-ポリヌクレオチド複合体には見られない特徴的な光学特性を有する産物-ポリヌクレオチド複合体。
(a)異なる光学特性を有する基質及び産物(多くの蛍光発生酵素基質が市販されており(例えば、Haugland, 1996参照)、グリコシダーゼ、ホスファターゼ、ペプチダーゼ、及びプロテアーゼの基質が含まれる(Craig et al., 1995、Huang et al., 1992、Brynes et al., 1982、Jones et al., 1997、Matayoshi et al., 1990、Wang et al., 1990))、又は
(b)類似する光学特性を有するが、基質ではなく産物のみがポリヌクレオチドに結合又は反応する基質及び産物。
(2)産物に特異的に結合又は反応し、これにより、ソーティングを可能にするポリヌクレオチドの光学特性の変化を誘導する試薬の追加(これらの試薬は、区画を破壊してポリヌクレオチドをプールする前又は後に添加することができる)。試薬は、
(a)基質及び産物の両方がポリヌクレオチドに付着している場合、基質ではなく産物に特異的に結合又は特異的に反応し、又は
(b)基質ではなく産物のみがポリヌクレオチドに結合又は反応する場合、基質及び産物の両方を任意に結合する。
(1)ポリヌクレオチドを発現させて、それぞれのポリペプチドを得るステップと、
(2)直接選択可能であってもなくてもよい産物への基質の転換を、所望の活性に応じて、ポリペプチドに触媒させるステップと、
(3)任意に、最初の反応を1つ以上の後続の反応に結合するステップであり、各反応は、前の反応の産物により調整され、最終的な選択可能な産物の作成につながるステップと、
(4)触媒作用の選択可能な産物をポリヌクレオチドに連結するステップであり、
a)産物がポリヌクレオチドと会合するような形で、基質をポリヌクレオチドに結合すること、又は
b)産物上に残る基質に付着した適切な分子「タグ」により、選択可能な産物をポリヌクレオチドと反応又は結合させること、又は
c)産物との産物特異的な反応又は相互作用により、(基質ではなく)選択可能な産物をポリヌクレオチドに結合させること、の何れかにより行われるステップと、
(5)触媒作用の産物を、その特徴的な光学特性により、又は産物に特異的に結合又は特異的に反応し、これによりポリヌクレオチドの光学特性に変化を誘導する試薬を添加することにより、それが結合したポリヌクレオチドと共に選択するステップと、を備え、ここでステップ(1)乃至(4)において、各ポリヌクレオチド及びそれぞれのポリペプチドは、マイクロカプセル内に含まれる。
(1)ポリヌクレオチドを発現させて、それぞれのポリペプチドを得るステップと、
(2)選択可能な分子の生成又は生存を可能にするような形で、所望の活性に応じて、ポリペプチドに生化学反応又は一連の共役反応を活性化又は阻害させるステップと、
(3)選択可能な分子をポリヌクレオチドに連結するステップであり、
a)選択可能な分子、又はそれが由来する基質を、ポリヌクレオチドに付着させること、又は
b)産物上に残る基質に付着した適切な分子「タグ」により、選択可能な産物をポリヌクレオチドに反応又は結合させること、又は
c)産物との産物特異的な反応又は相互作用により、(基質ではなく)触媒作用の産物をポリヌクレオチドに結合させること、又は
d)反応の成分と任意の産物とをゲル内に固定化すること(このモードにおいて、プロセスの開始時にカプセル化された反応成分にゲル形成剤を含める必要があり、ゲルは、例えば、反応をゲルの相変化温度まで冷却することにより形成され得る)、又は
e)反応成分と任意の産物とをマイクロカプセル内に維持すること、の何れかにより行われるステップと、
(4)選択可能な産物を、その特徴的な光学特性により、又は産物に特異的に結合又は特異的に反応し、これによりポリヌクレオチドの光学特性に変化を誘導する試薬を添加することにより、それが結合したポリヌクレオチドと共に選択するステップと、を備え、ここでステップ(1)乃至(3)において、各ポリヌクレオチド及びそれぞれのポリペプチドは、マイクロカプセル内に含まれる。
(1)定評のある製造業者(例えば、Becton-Dickinson、Coulter)の市販の蛍光活性化細胞ソーティング設備により、1時間当たり最大108個のビーズ、カプセル、ポリヌクレオチド、及び/又はポリペプチド(イベント)をソーティングすることが可能であり、
(2)各ビーズからの蛍光シグナルは、ビーズに付着した蛍光分子の数と緊密に対応しており、現在、粒子当たり僅か数百個の蛍光分子を定量的に検出可能であり、
(3)蛍光検出器の広いダイナミックレンジ(一般に4log単位)により、ソーティング手順のストリンジェンシを容易に設定できるため、開始プールから最適な数のビーズ、カプセル、ポリヌクレオチド、及び/又はポリペプチドを回収することが可能であり(ゲートは、蛍光の小さな差でビーズを分離するように、又は蛍光の大きな差でのみビーズを分離するように、実行される選択に応じて設定することが可能)、
(4)市販の蛍光活性化細胞ソーティング設備は、2種類までの異なる波長で同時励起を行い、4種類までの異なる波長で蛍光を検出することが可能であり(Shapiro, 1983)、2種類(以上)の異なる蛍光マーカを備えたビーズ、カプセル、ポリヌクレオチド、及び/又はポリペプチドの標識をモニタすることにより、正及び負の選択を同時に実行することができ、例えば、酵素の2種類の代替基質(例えば2つの異なるエナンチオマ)が異なる蛍光タグにより標識されている場合、ビーズ、カプセル、ポリヌクレオチド、及びポリペプチドは、使用される基質に応じて異なるフルオロフォアにより標識可能であり、エナンチオ選択性を有する酵素をコードする遺伝子のみが選択され、
(5)高均一性の誘導体化/非誘導体化非磁性/常磁性微粒子(ビーズ)は、多くの供給元(Sigma、Molecular Probes等)から市販されている(Fornusek and Vetvicka, 1986)。
Aqueous: 水溶液
Oid/surfactant: 油/界面活性剤
Nozzel (5μm deep): ノズル(深さ5μm)
Flow channel (25μm deep): 流路(深さ25μm)
Femto-droplets: フェムト液滴
[図2A乃至2D]
Oid/surfactant: 油/界面活性剤
Femtodroplets: フェムト液滴
Nozzel, 5μm deep: ノズル(深さ5μm)
Flow channel, 25μm deep: 流路(深さ25μm)
Normalized droplet count: 正規化液滴数
μL/h oil: μL/h 油
Droptet diameter: 液滴径
[図3]
Droplet diameter: 液滴径
Oil flow rate: 油流量
Volume: 体積
Droplet generation rate: 液滴生成速度
[図4]
Emulsion filled syringe: エマルション充填シリンジ
Syringe needle: シリンジ針
Microfluidic device: マイクロ流体デバイス
[図5A]
Water-in-oil droplet generation: 油中水型液滴生成
Aq: 水溶液
Oil: 油
44,000 droplets per second: 毎秒44,000液滴
Collection: 収集
Agarose bead-in-water-in-oil droplet generation: 油中水中アガロース型液滴生成
Aqueout phase: 水相
[図5B]
Primary emulsion (water-in-oil) droplets: 一次エマルション(油中水型)液滴
Aqueous inner phase: 水性内相
External aqueous phase with surfactant: 界面活性剤を含む外部水性相
Hydrophilic channels: 親水性チャネル
Organic middle phase: 有機中相
Inlet: 入口
Outlet: 出口
Aqueous inlet: 水溶液入口
Debris filter: デブリフィルタ
Oil inlet: 油入口
Emulsion inlet: エマルション入口
Flow-focusing junction: フローフォーカシング接合部
Oil: 油
Aq: 水溶液
Emulsion: エマルション
OD=外形
ID=外形
[図7A乃至7D]
Side scatter: 側方散乱
Detection threshold: 検出閾値
Monodisperse droplets: 単分散液滴
Occupied: 占有
Unoccupied: 未占有
Forward scatter: 前方散乱
Green fluorecence intensity: 緑色蛍光強度
Monodisperse oil-in-water droplets: 単分散水中油型液滴
[図8A]
Agarose-in-oil droplet generation: 油中アガロース型液滴生成
2% agarose & DNA: 2%アガロース及びDNA
Phi29 DNA polymerase: Phi2 DNAポリメラーゼ
Oil: 油
44,000 droplets per second: 毎秒44,000液滴
External Organic phase: 外部有機相
30 ℃ incubation: 30℃でインキュベーション
Break emulsion: 解乳化
Amplified DNA: 増幅DNA
Agarose bead: アガロースビーズ
Agarose bead with Phi29 DNA polymerase amplified DNA: Phi2 DNAポリメラーゼ増幅DNAを有するアガロースビーズ
[図8B及び8C]
Agarose-in-IVTT-in-oil droplet generation: 油中IVTT中アガロース型液滴生成
Agarose beads: アガロースビーズ
Oil: 油
4,000 droplets per second: 毎秒4,000液滴
IVTT phase: IVTT相
Surfactant layer: 界面活性剤層
External oil phase: 外部油層
Agarose-in-IVTT-in-oil-in-water droplet generation: 水中油中IVTT中アガロース型液滴生成
Aq: 水溶液
Agarose inner phase with amplified DNA: 増幅DNAを有するアガロース内相
Read out: 読み取り
Flow cytometry: フローサイトメトリ
Microscopy: 顕微鏡
Oil shell: オイルシェル
External aqueous phase: 外部水相
[図9A]
Microfluidic device: マイクロ流体デバイス
Fluidic tubing: 流体チューブ
Glass syringes: ガラスシリンジ
Heating pad: 過熱パッド
Syringe pumps: シリンジポンプ
[図9B]
Phi29 + hexamers: Phi29+六量体
Surfactant + oil: 界面活性剤+油
2 % agarose + DNA: 2%アガロース+DNA
Electric heater: 電気ヒータ
[図10A]
Phi29 + hexamers: Phi29+六量体
2% agarose + DNA: 2%アガロース+DNA
Nozzel (5μm deep): ノズル(深さ5μm)
Femto-droplets: フェムト液滴
Flow channel (25μm deep): 流路(深さ25μm)
Agarose bead: アガロースビーズ
[図10B]
Normalized bead count: 正規化ビーズ数
Bead diameter: ビーズ径
[図10C]
Unwashed: 未洗浄
Fluorescence: 蛍光
Merge: 融合
[図11A乃至11D]
Count: カウント
Control: 対照
DNA alone: DNAのみ
Green fluorescence intensity: 緑色蛍光強度
Single-molecule encapsulation: 単一分子カプセル化
[図13A]
Phi29 DNA amplification of plasmid DNA in agarose beads: アガロースビーズにおけるプラスミドDNAのPhi29 DNA増幅
Side scatter: 側方散乱
Agarose beads: アガロースビーズ
Forward scatter: 前方散乱
Count: カウント
Negative control: 陰性対照
Green fluorescence intensity: 緑色蛍光強度
[図13B]
IVTT of GFP from Phi29 amplified DNA in droplets: 液滴におけるPhi29増幅DNAからのGFPのIVTT
Side scatter: 側方散乱
Singly occupied: 単一占有
Unoccupied droplets: 未占有液滴
Forward scatter: 前方散乱
Agarose beads: アガロースビーズ
TAE alone: TAEのみ
Green fluorescence intensity: 緑色蛍光強度
[図14]
SICLOPPS intein: SICLOPPSインテイン
active intein: 活性インテイン
cyclic peptide: 環状ペプチド
lariat: ラリアット
thioester: チオエステル
[図15]
LacI coding sequence: LacIコード配列
f1 origin: f1開始点
bla (Ap) coding sequence: bla (Ap) コード配列
pBR322 origin: pBR322開始点
C-intein: Cインテイン
Library: ライブラリ
N-intein: Nインテイン
[図16]
Intensity: 強度
CLLFVY in IVTT bulk: IVTTバルクにおけるCLLFVY
CLLFVY IVTT post FACS sorting: CLLFVY IVTT FACSソーティング後
[図17]
Mormalized to Mode: モードに正規化
Buffer alone: バッファのみ
IVTT alone: IVTTのみ
Green fluorescence intensity: 緑色蛍光強度
[図18A]
Normalized to Mode: モードに正規化
Single emulsion: シングルエマルション
Buffer alone: バッファのみ
IVTT alone: IVTTのみ
TX4 library: TX4ライブラリ
Green fluorescence intensity: 緑色蛍光強度
[図18B]
Side scatter: 側方散乱
Double emulsions: ダブルエマルション
Forwad scatter: 前方散乱
Claims (17)
- 環状ポリペプチドと前記環状ポリペプチドをコードするポリヌクレオチドとを共区画化する方法であって、
a)前記環状ポリペプチドをコードするポリヌクレオチドを含む区画を形成するステップと、
b)前記ポリヌクレオチドからポリペプチドを発現させるステップと、
c)前記ポリペプチドを環化させるステップと、を備える方法。 - 環状ポリペプチドをソーティングする方法であって、請求項1のステップを備え、更に
c)前記環状ポリペプチドを活性についてスクリーニングするステップと、
d)所望の活性を示す前記環状ポリペプチドを選択するステップと、を備える方法。 - 更に、前記ポリヌクレオチドを増幅するステップを備える、請求項1又は2記載の方法。
- 前記区画は、更に、ゲル形成剤を含み、前記ゲル形成剤は、前記ポリヌクレオチドを増幅した後、固化させてゲルビーズにする、請求項3記載の方法。
- 前記区画は、前記ゲル形成剤を固化させてゲルビーズにした後に、破壊される、請求項4記載の方法。
- 前記ゲルビーズは、前記環状ポリペプチドを発現させるための条件下に置かれる、請求項5記載の方法。
- 新たな区画が、前記ゲルビーズの周りに形成される、請求項5又は6記載の方法。
- 前記ポリヌクレオチドは、N末端インテイン断片をコードする配列と、それに続く前記環状ポリペプチドをコードする配列と、それに続くC末端インテイン断片をコードする配列とを含む、先行請求項の何れかに記載の方法。
- 前記区画は、水中油中水型(w/o/w)エマルションの液滴、小胞、又は区画である、先行請求項の何れかに記載の方法。
- 前記ポリヌクレオチドから前記ポリペプチドを発現させることは、前記ポリヌクレオチドを、非天然アミノ酸をチャージした1つ以上のtRNAを含むIVTT混合物と接触させるステップを含む、先行請求項の何れかに記載の方法。
- 増幅は、容器内で実行され、熱は、前記容器を取り囲む表面積全体で均一且つ連続的に加えられる、請求項4乃至10の何れかに記載の方法。
- a)N末端インテイン断片をコードする配列と、それに続く環状ポリペプチドをコードする配列と、それに続くC末端インテイン断片をコードする配列とを含むポリヌクレオチドと
b)前記環状ペプチドと、を備える区画。 - 請求項12記載の複数の区画を備える環状ポリペプチドのライブラリ。
- a)マイクロ流体デバイスと、
b)N末端インテイン断片をコードするポリヌクレオチドと、
c)C末端インテイン断片をコードするポリヌクレオチドと、を備えるキット。 - 更に、カプセル形成材料を備える、請求項14記載のキット。
- 前記カプセル形成材料は、油、脂質、又は高分子電解質を含む、請求項15記載のキット。
- 更に、ゲル形成剤を備える、請求項14から16の何れか記載のキット。
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JP2003534768A (ja) * | 1998-12-18 | 2003-11-25 | ザ ペン ステート リサーチ ファウンデーション | 環状ペプチド |
JP2008515421A (ja) * | 2004-10-08 | 2008-05-15 | メディカル リサーチ カウンシル | マイクロ流体システム内のインビトロ進化 |
WO2012156744A2 (en) * | 2011-05-17 | 2012-11-22 | Cambridge Enterprise Limited | Gel beads in microfluidic droplets |
Also Published As
Publication number | Publication date |
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EP3586134B1 (en) | 2023-12-13 |
KR102567657B1 (ko) | 2023-08-16 |
US20200049695A1 (en) | 2020-02-13 |
WO2018154021A1 (en) | 2018-08-30 |
ZA201905538B (en) | 2024-01-31 |
AU2018223899C1 (en) | 2024-03-28 |
MX2019010007A (es) | 2019-12-19 |
AU2018223899A1 (en) | 2019-09-12 |
AU2018223899B2 (en) | 2023-11-30 |
CA3054251A1 (en) | 2018-08-30 |
EP3586134C0 (en) | 2023-12-13 |
ES2972599T3 (es) | 2024-06-13 |
CN110520729A (zh) | 2019-11-29 |
EP3586134A1 (en) | 2020-01-01 |
CN110520729B (zh) | 2024-05-28 |
GB201702938D0 (en) | 2017-04-12 |
KR20190137078A (ko) | 2019-12-10 |
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