JP6281953B2 - 核酸分子の酸化還元活性を評価する方法および酸化還元活性を有する核酸分子 - Google Patents
核酸分子の酸化還元活性を評価する方法および酸化還元活性を有する核酸分子 Download PDFInfo
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Description
前記デバイスが、検出部を備える基板を有し、前記検出部が、電極系を有し、前記基板上に、前記評価対象の核酸分子が配置されていることを特徴とする。
本発明の評価方法は、前述のように、酸化還元反応を電気化学的に検出するデバイスを用いて、評価対象の核酸分子により触媒される、基質に対する酸化還元反応を、電気化学的に検出する検出工程と、
前記酸化還元反応の検出結果から、前記核酸分子の酸化還元活性を評価する評価工程とを含み、
前記デバイスが、検出部を備える基板を有し、
前記検出部が、電極系を有し、
前記基板上に、前記評価対象の核酸分子が配置されていることを特徴とする。
本発明の第1実施形態においては、前述のように、前記基板上に、前記評価対象の核酸分子を配置したデバイスを使用する。
本発明の第2実施形態においては、前述のように、前記基板上に、前記アプタマーが結合された前記評価対象の核酸分子を配置したデバイスを使用する。特に示さない限りは、前記第1実施形態と同様である。
本発明のスクリーニング方法は、前述のように、前記本発明の評価方法により、デバイスを用いて評価対象の核酸分子の酸化還元活性を評価し、酸化還元活性を有する核酸分子をスクリーニングすることを特徴とする、酸化還元活性を有する核酸分子のスクリーニング方法である。
本発明の酸化還元活性を有する核酸分子は、下記(a)〜(d)からなる群から選択された少なくとも一つのポリヌクレオチドを含むことを特徴とする。
(a)配列番号1〜132のいずれかの塩基配列からなるポリヌクレオチド
(b)前記(a)のいずれかの塩基配列において、1もしくは数個の塩基が欠失、置換、挿入および/または付加された塩基配列からなり、酸化還元活性を有するポリヌクレオチド
(c)前記(a)のいずれかの塩基配列に対して、80%以上の同一性を有する塩基配列からなり、酸化還元活性を有するポリヌクレオチド
(d)前記(a)のいずれかの塩基配列からなるポリヌクレオチドに対してストリンジェントな条件下でハイブリダイズするポリヌクレオチドに、相補的な塩基配列からなり、酸化還元活性を有するポリヌクレオチド
A.条件検討
評価対象の核酸分子の酸化還元反応を検出するにあたって、その条件を検討した。
以下に示すように、市販の電気化学検出型マイクロアレイ(商品名CombiMatrix ElectraSense microarray、CombiMatrix社製)における電極上に、スペーサーを介して、既知のポリヌクレオチドを固定化した。
EAD2(配列番号133)
CTGGGAGGGAGGGAGGGA
SA(配列番号134)
CCGACGCACCGATCGCAGGTTCGG
前記マイクロアレイに、基質として過酸化水素を添加し、酸化還元反応により生じる電気シグナルを電流として測定した。測定は、測定装置(製品名ElectraSense Reader、CombiMatrix社)を使用した(以下、同様)。前記過酸化水素は、所定濃度(0、1、2、4、8mmol/L)となるように各種緩衝液に添加し、基質溶液として添加した。前記緩衝液は、トリス緩衝液(pH7.4)、トリス緩衝液(pH8.0)、トリス緩衝液(pH8.5)、トリス緩衝液(pH9.0)を使用し、それぞれ、過酸化水素添加後のpHが、かっこ内の値となるように調整した。
前記「A.条件検討」と同じ条件で、前記スペーサーの長さが異なる4種類のEAD2(dT0、dT8、dT16、dT24)各250個について、酸化還元反応の測定を行った。
前記「A.条件検討」における、前記スペーサーの長さが異なる4種類のEAD2(dT0、dT8、dT16、dT24)各250個の測定結果、および、前記スペーサーの長さが異なる4種類のSA(dT0、dT8、dT16、dT24)各250個の測定結果について、正規分布に従っているか否かを確認した。また、Kolmoforov−Smirnov検定によりP値を求めた。
異なる配列からなる複数のポリヌクレオチドをマイクロアレイチップに固定化し、酸化還元反応の測定を行った。
以下に示すように、市販のマイクロアレイチップ(商品名CustomArray(登録商標)12K、CombiMatrix社製)における電極上に、24塩基長のポリdTからなるスペーサーを介して、複数のポリヌクレオチドを固定化した。
EAD2(配列番号133)
CTGGGAGGGAGGGAGGGA
SA(配列番号134)
CCGACGCACCGATCGCAGGTTCGG
c−Myc(配列番号135)
TGAGGGTGGGGAGGGTGGGGAA
TA(配列番号136)
GGTTGGTGTGGTTGG
前記「A.再現性」において得られた測定結果に基づいて、図5(A)〜(D)のグラフに、(A)c−Myc 100個のシグナル値とその頻度との関係、(B)SA 100個のシグナル値とその頻度との関係、(C)EAD2 100個のシグナル値とその頻度との関係、(D)TA 100個のシグナル値とその頻度との関係を示した。いずれにおいても、シグナルの分布は、正規分布に従っていることが確認できた。
前記実施例2のマイクロアレイチップを用いて、高い酸化還元活性を有する新たなDNAzymeをスクリーニングした。
Claims (2)
- 配列番号2、7、8、10、11、13−15、17、20−23、25、27−29、31、33、34、38−40、42−46、48−53、55、56、58、61、62、65、69、71、74、76、79、82−85、88−93、99、102−109、118、126、127、および132からなる群から選択された少なくとも一つのポリヌクレオチドを含むことを特徴とする酸化還元活性を有するDNAzyme。
- 配列番号15、22、33、35、39、50、55、65、76、83、90、97、105および108からなる群から選択された少なくとも一つのポリヌクレオチドを含むことを特徴とする酸化還元活性を有する核酸分子。
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WO2014136560A1 (ja) * | 2013-03-08 | 2014-09-12 | Necソリューションイノベータ株式会社 | 核酸素子候補分子、および、これを用いたターゲット分析用核酸素子のスクリーニング方法 |
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WO2007047986A1 (en) * | 2005-10-21 | 2007-04-26 | Wisconsin Alumni Research Foundation | Method and system for delivering nucleic acid into a target cell |
US8569468B2 (en) | 2006-09-14 | 2013-10-29 | The Regents Of The University Of California | Nanoplasmonic molecular ruler for nuclease activity and DNA footprinting |
CN104651381A (zh) | 2008-01-03 | 2015-05-27 | 巴斯夫酶有限责任公司 | 转移酶和氧化还原酶、编码它们的核酸以及其制备和应用方法 |
JP2010011791A (ja) | 2008-07-03 | 2010-01-21 | Toshiba Corp | 複数核酸の検出方法 |
WO2010142037A1 (en) | 2009-06-08 | 2010-12-16 | The University Of Western Ontario | An electrochemical method and apparatus of identifying the presence of a target |
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EP2730915A4 (en) | 2015-06-17 |
EP2730915A1 (en) | 2014-05-14 |
CN103649741A (zh) | 2014-03-19 |
EP2730915A9 (en) | 2015-03-25 |
CN104388424B (zh) | 2017-07-07 |
HK1204653A1 (en) | 2015-11-27 |
US9637737B2 (en) | 2017-05-02 |
CN104388424A (zh) | 2015-03-04 |
US20140128589A1 (en) | 2014-05-08 |
WO2013005723A1 (ja) | 2013-01-10 |
JP5863794B2 (ja) | 2016-02-17 |
US20160060630A1 (en) | 2016-03-03 |
CN103649741B (zh) | 2016-11-16 |
JPWO2013005723A1 (ja) | 2015-02-23 |
US10138480B2 (en) | 2018-11-27 |
BR112013033443A2 (pt) | 2017-01-31 |
JP2016028255A (ja) | 2016-02-25 |
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