DK2569425T3 - Endoribonuklease-sammensætninger og fremgangsmåder til anvendelse deraf - Google Patents

Endoribonuklease-sammensætninger og fremgangsmåder til anvendelse deraf Download PDF

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DK2569425T3
DK2569425T3 DK11781086.1T DK11781086T DK2569425T3 DK 2569425 T3 DK2569425 T3 DK 2569425T3 DK 11781086 T DK11781086 T DK 11781086T DK 2569425 T3 DK2569425 T3 DK 2569425T3
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Rachel E Haurwitz
Jennifer A Doudna
Blake Wiedenheft
Martin Jinek
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Univ California
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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Claims (23)

1. Csy4-endoribonukleasevariant omfattende en aminosyresekvens, der har mindst ca. 95 % aminosyresekvensidentitet med aminosyresekvensen med SEQ ID NO:101 eller SEQ ID NO: 102 angivet i figur 6, hvor endoribonuklea-sen omfatter en aminosyresubstitution ved His-29, hvor Csy4-endoribonukleasevarianten er enzymatisk inaktiv i fravær af imidazol, og hvor Csy4-endoribonukleasevarianten er aktiverbar i nærvær af imidazol.
2. Csy4-endoribonukleasevariant ifølge krav 1, hvor aminosyresubstitutionen er en His29- til Ala29-substitution.
3. Csy4-endoribonukleasevariant ifølge krav 1, hvor Csy4- endoribonukleasevarianten omfatter en del, der tilvejebringer et detekterbart signal.
4. Csy4-endoribonukleasevariant ifølge krav 3, hvor delen er udvalgt blandt en fluorophor, et kvantepunkt, et enzym, som ikke er endoribonukleasen, eller en nanopartikel.
5. Csy4-endoribonukleasevariant ifølge krav 1, hvor endoribonukleasen er immobiliseret på en uopløselig bærer.
6. Csy4-endoribonukleasevariant ifølge krav 5, hvor den uopløselige bærer er en kugle.
7. Csy4-endoribonukleasevariant ifølge krav 1, hvor Csy4- endoribonukleasevarianten binder et RNA-substrat omfattende nukleotidse-kvensen 5'-GUUCACUGCCGUAUAGGCAGCUAAGAAA-3' (SEQ ID NO:1).
8. Nukleinsyre omfattende en nukleotidsekvens, der koder for Csy4-endoribonukleasevarianten ifølge krav 1.
9. Rekombinant ekspressionsvektor omfattende en nukleotidsekvens, der koder for Csy4-endoribonukleasevarianten ifølge krav 1.
10. Rekombinant ekspressionsvektor ifølge krav 9, hvor nukleotidsekvensen, der koder for Csy4-endoribonukleasevarianten er funktionsmæssigt forbundet med en promoter.
11. Rekombinant ekspressionsvektor ifølge krav 10, hvor promoteren er en inducerbar promoter.
12. In vitro genmodificeret værtscelle omfattende den rekombinante ekspressionsvektor ifølge krav 9.
13. Kit til rensning af et target-RNA, der er til stede i en blandet population af nukleinsyrer, hvilket kit omfatter: Csy4-endoribonucleasevarianten ifølge krav 1.
14. Kit ifølge krav 13, hvor endoribonukleasen er immobiliseret på en uopløselig bærer.
15. Kit ifølge krav 13 eller 14, yderligere omfattende en rekombinant ekspressionsvektor omfattende, i rækkefølge fra 5' til 3' og i funktionsmæssig binding: a) en nukleotidsekvens, der koder for et RNA-substrat, der er specifikt bundet af Csy4-endoribonukleasevarianten ifølge krav 1; b) et multipelt kloningssted, der er egnet til insertion af en nukleinsyre, der koder for target-RNA'et.
16. Kit ifølge krav 15, hvor nukleotidsekvensen, der koder for RNA-substratet, er funktionsmæssigt forbundet med en promoter.
17. Kit ifølge krav 16, hvor promoteren er en inducerbar promoter.
18. Kit ifølge krav 15, hvor RNA-substratet omfatter nukleotidsekvensen 5'-GUUCACUGCCGUAUAGGCAGCUAAGAAA-3' (SEQ ID NO:1).
19. Kit ifølge krav 15, hvor den rekombinante ekspressionsvektor omfatter, insereret i det multiple kloningssted, en nukleotidsekvens, der koder for tar- get-RNA'et.
20. Kit ifølge krav 13, yderligere omfattende en eller flere af (a) imidazol, (b) en eller flere vaskebuffere og (c) en positiv kontrol-ekspressionsvektor.
21. Fremgangsmåde til isolering af et target-RNA, der er til stede i en blandet population af nukleinsyrer, hvilken fremgangsmåde omfatter: a) at bringe en blandet population af nukleinsyrer i kontakt med Csy4-endoribonukleasevarianten ifølge krav 1, hvor Csy4-endoribonukleasevarianten er immobiliseret på en uopløselig bærer, hvor den blandede population af nukleinsyrer omfatter et mærket target-RNA omfattende en genkendelsesnukleotidsekvens, der er specifikt bundet af den immobiliserede Csy4-endoribonukleasevariant, hvorved der dannes et mærket target-RNA-immobiliseret Csy4-endoribonukleasevariant-kompleks, hvor det at bringe i kontakt udføres i en bindingsopløsning uden imidazol; b) at tilsætte imidazol til bindingsopløsningen til en endelig koncentration på fra ca. 100 mM til ca. 500 mM, hvorved der dannes en reaktiveringsopløsning, som enzymatisk reaktiverer den immobiliserede Csy4-endoribonukleasevariant, hvor den reaktiverede immobiliserede Csy4-endoribonukleasevariant spalter target-RNA'et fra markøren; og c) at indsamle det frigjorte target-RNA.
22. Fremgangsmåde ifølge krav 21, yderligere omfattende et vasketrin udført efter trin (a) og før trin (b).
23. Fremgangsmåde til isolering af et polypeptid, der binder et target-RNA, hvilken fremgangsmåde omfatter: a) at bringe et immobiliseret kompleks i kontakt med en flydende opløsning omfattende et polypeptid, der binder target-RNA'et, hvor det immobiliserede kompleks omfatter Csy4-endoribonukleasevarianten ifølge krav 1 og et mærket target-RNA, der omfatter en genkendelsesnukleotidsekvens, der er specifikt bundet af Csy4-endoribonukleasevarianten, hvor det at bringe i kontakt resulterer i binding af polypeptidet til target-RNA'et, hvor det at bringe i kontakt udføres i en bindingsopløsning uden imidazol; og b) at eluere det bundne polypeptid.
DK11781086.1T 2010-05-10 2011-05-09 Endoribonuklease-sammensætninger og fremgangsmåder til anvendelse deraf DK2569425T3 (da)

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US33316310P 2010-05-10 2010-05-10
US36562710P 2010-07-19 2010-07-19
US41328710P 2010-11-12 2010-11-12
PCT/US2011/035775 WO2011143124A2 (en) 2010-05-10 2011-05-09 Endoribonuclease compositions and methods of use thereof

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US20150284697A1 (en) 2015-10-08
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JP5926242B2 (ja) 2016-05-25
EP2569425A2 (en) 2013-03-20
EP2569425B1 (en) 2016-07-06
EA201291024A1 (ru) 2013-11-29

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