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KR900700603A - 리보자임 - Google Patents

리보자임

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KR900700603A
KR900700603A KR1019890701528A KR890701528A KR900700603A KR 900700603 A KR900700603 A KR 900700603A KR 1019890701528 A KR1019890701528 A KR 1019890701528A KR 890701528 A KR890701528 A KR 890701528A KR 900700603 A KR900700603 A KR 900700603A
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ribozyme
rna
target rna
sequence
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필립 헤이스로프 제임스
라일 게르라히 웨인
안토니 제닝스 필립
헬렌 카메론 피오나
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알프레드 페르난트
컴온웰스 사이언티픽 엔드 인더스트리얼 리서어치 오르 가니제이숀
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Abstract

내용 없음

Description

리보자임
본 내용은 요부공개 건이므로 전문내용을 수록하지 않았음
제1도는 야생주와 변이된 RNA의 RNA지체 절단 부위; 및 자체 촉매된 RNA절단 생성물이 나타내는 전기영동의 프로파일을 나타내고 있다. (a) ASBV, 영원 수반 DNA전사물 및 sTobRV, LTSV, VMOV(벨벳 담배의 반점 비루스), SNMV(솔라늄 노디플로륨반점 비루스) 및 SCMoV(서브데라닌 클로버 반점 비루스)의수반, RNA의 자연 발생의 RNA절단 부위와 연관된 보유 구조를 요약함. 상기 구조사이에 보유된 뉴클레오티드 서열을 나타내었으며, 그 이외는 X로 표시하였다. 염기쌍은 "＊"로 나타내었고 RNA 절단 부위는 화살표시를 하였다. (b) sTobRV RNA의 (+)가닥 절단과 관련된 보유 뉴클레오티드 서열을 나타냄. 절단 부위는 화살표시로 하였다. (c) 3개의 뉴클레오티드(UGU 잔기 7 내지 9)의 플랭킹 복제와 함께 8개 뉴클레오티드(박스로 표시)의 삽입을 비롯한 sTobRV의 생체밖 변이주를 나타내었다. (d) sTobRV야생주 및 생체밖 변이주 D-51의 서브클로운 Hae Ⅲ 단편을 (+)아 (-) 두 배향으로 각각 전사하고 방사선 표지된 전사물을 폴리아크릴아미드 겔 전기영동으로 분리한다. 야생주(WT)와 변이주(D-51) 서열로부터 미 절단의 159와 170염기의 위치가 전사된 것을 나타낸다.
제2도는 리보자임의 뉴클레오티드 서열과 겔 전기영동에 의해 분리된 리보자임 절단의 생성물을 나타내고 있다. (a) D-51변이주의 삽입된 뉴클레오티드(제1c도)는 HamHI제한 효소 부위를 포함한다. BamHI은 변이주 DNA를 자르는데 사용하며 두 서열은 서브 클로운되고 생체 밖에서 각각 전사된다. RNA전사는 "＊"로 표시된 RNA간에 강력한 염기쌍을 도식적으로 나타내었다. 화살표시된 절단 부위를 포함한 단편은 S-RNA로 정의하며, 리보자임을 포함한 단편은 R2-RNA로 정의한다. (b) [32P]-R2-RNA(101염기)를 단독(레인1) 및 비 표지된 S-RNA와 함께(레인2) 배양하였다. [32P]-S2-RNA를 단독(레인3) 및 비 표지된 것과32P표지된 R2-RNA(각각 레인 4 및 5)과 함께 배양한다.
제3도는 본 발명의 한 태양에 따른 리보자임의 도식적인 모델을 나타낸다. A영역은 타겟 RNA내에 절단 서열을 나타내며 B영역은 촉매 영역을, C영역은 리보자임의 아암을 나타낸다.

Claims (24)

  1. 적어도 타켓 RNA의 일부분에 대한 뉴클레오티드 서열에 상보적인 하이브리다이즈 영역과 이 하이브리다이즈 영역이 9개 이상의 뉴클레오티드를 포함하는 상기 타겟 RNA를 절단시킬 수 있는 촉매 영역을 포함한 리보자임(ribozyme).
  2. 적어도 타켓 RNA의 일부분에 대해 상보적인 서열을 가지며, 단일 가닥의 RNA로 형성된 하나이상의 아암(arm)을 포함하며, 이 하나이상의 아암이 상기 타겟 RNA를 절단할 수 있는 촉매 영역과 관련되어 있으며; 하이브리다이즈 영역이 RNA의 단일 아암을 포함하는 경우, 상기 아암은 적어도 9개 뉴클레오티드를 포함하며, 하이브리다아즈 영역이 2개 이상의 RNA아암을 포함하는 경우, 상기 아암중 뉴클레오티드의 합이 9이상인 리보자임.
  3. 제2항에 있어서, 상기 촉매 영역 전반에 걸쳐 서로 연관된 2개의 RNA단일 가닥의 아암을 갖는 리보자임.
  4. 전술한 항중 어느 한항에 있어서, 절단된 타켓 RNA를 변형시키지 않는 리보자임.
  5. 하기식(1)의 리보자임:
    상기식에서, X는 임의의 리보뉴클레오티드를 나타내며 각 X 잔기는 같거나 상이하며; n과 n′의 합은 6이상이며, n과 n′은 같거나 상이하며; (＊)는 상보적인 리보뉴클레오티드 간의 염기 쌍을 나타내며; X′과 X″은 올리고 리보뉴클레오티드 간에 염기쌍을 형성할 수 있도록 그 길이의 적어도 일부분에 상보적 서열의 리보뉴클레오티드를 나타내거나 X′와 X″이 함께 단일의 RNA서열을 형성하여 상기 서열중 적어도 일부분이 상보적인 뉴클레오티드간의 염기쌍을 형성한 가지를 포함하며; 선택적으로, A,G,C 또는 U중 어느 하나로부터 선택된 부가의 뉴클레오티드가1A이후에 삽입됨.
  6. 전술한 항중 어느 한 항에 있어서, 하기식(2)을 갖는 리보자임.
    상기식에서, X는 임의의 리보뉴클레오티드를 나타내며 각 X 잔기는 같거나 상이하며;(＊)는 상보적인 리보뉴클레오티드 간의 염기 쌍을 나타내며; n과 n′은 상기 규정한 바와 같으며; B는 결합, 염기 쌍, 리보뉴클레오티드 또는 적어도 2개의 리보뉴클레오티드를 포함한 올리고 리보뉴클레오티드를 나타내며; m과 m′은 1이 상이며 같거나 상이하고; 및 선택적으로, A,G,C 또는 U중 어느 하나로부터 선택된 부가의 뉴클레오티드가1A이후에 삽입됨.
  7. 제5항 또는 제6항에 있어서, n과 n′의 합이 16이상인 리보자임.
  8. 전술한 항중 어느 한항에 있어서, XO가 리보뉴클레오티드이고, U는 우라실이며 Y는 아데닌, 시토신 또는 우라실인 서열 XOUY 뒤의 타켓 RNA를 절단할 수 있는 촉매 영역이 있는 리보자임.
  9. 각 플랭킹(flanking)서열이 불활성화 시키는데 필요한 타켓 RNA에 상보적인 서열을 포함하는 플랭킹 서열 전체에 연결되어 있는 다수의 촉매 분역을 포함한 리보자임.
  10. 전술한 항중 어느 한항에 있어서, 원형화에 의해 안정된 리보자임.
  11. 전술한 항중 어느 한항에 있어서, 촉매성 역감작 서열을 포함한 리보자임.
  12. 타켓 RNA를 제1항 내지 제11항중 어느 한 항에 따른 리보자임과 반응시키는 것을 포함한 상기 타켓 RNA의 불활성화 또는 절단방법.
  13. 제12항에 있어서, 진핵 또는 원핵 세포나 그것의 전구체로 트랜스펙트(transfect)된 DNA 또는 RNA전이 벡터로부터 상기 리보자임을 전사시키거나 숙주 게놈으로 결합시켜서, 그 결과, 상기 리보자임이 요구되는 세포의 타겟 RNA에 하이브리다이즈되어 불활성화 시킴으로써, 상기 불활성화가 원핵 또는 진핵 세포의 생체내에서 발생하는 방법.
  14. 제13항에 있어서, 상기 세포가 식물 또는 동물세포인 방법.
  15. 제13항 또는 제14항에 있어서, 타겟 RNA가 세포에 대해 내인성이거나 비루스성 RNA같이 외인성인 방법.
  16. 제15항에 있어서, 타겟 비루스 RNA가 HIV(HIDS), 간염 또는 포유류의 질병을 야기시키는 기타 비루스의 RNA로부터 선택되는 방법.
  17. 제15항에 있어서, 비루스성 RNA가 박테리오파지 RNA인 방법.
  18. 제1항 내지 제11항중 어느 한항의 리보자임의 유효량을 상기의 치료가 필요한 환자에게 투여하여, 상기 리보자임이 상기 비루스성 RNA에 하이브리다이즈 하여 그것의 전사를 불활성화 시킴으로서 인간이나 동물의 비루스성 질병을 치료하는 방법.
  19. 제1항 내지 제11항중 어느 한항의 리보자임을 식물에 투여하여, 상기 리보자임이 상기 비루스의 RNA에 하이브리다이즈하고 그것의 전사를 불활성화시키는 것을 포함한 식물의 비루스 또는 비로이드 질병을 치료하는 방법.
  20. 제1항 내지 제11항중 어느 한항의 리보자임을 약제학적, 수의학적 또는 농업적 허용담체 또는 부형제와 관련하여 포함하는 조성물.
  21. (a) 상기 리보자임에 상응하는 뉴클레오티드 서열을 DNA, RNA 또는 그것의 배합물을 포함한 전이 벡터로 결찰시키고; (b) 단계 (a)의 뉴클레오티드 서열을 RNA폴리머라제로 전사하고; 및 (c) 선택적으로 리보자임을 정제하는 단계를 포함한 제1항 내지 제11항중 어느 한항에 의한 리보자임을 생성하는 방법.
  22. 전사시 제1항 내지 제11항중 어느 한항이 리보자임을 산출하는 뉴클레오티드 서열을 포함한 RNA 또는 DNA 또는 그것의 배합물로 구성된 전이벡터.
  23. 제22항에 있어서, 박테리아 플라즈미드나 파지 DNA인 전이 벡터.
  24. 전사시 제1항 내지 제11항중 어느 한항의 리보자임을 산출하는 뉴클레오티드 서열을 포함한 원핵 또는 진핵 세포.
    ※ 참고사항 : 최초출원 내용에 의하여 공개하는 것임.
KR1019890701528A 1987-12-15 1988-12-14 올리고리보뉴클레오티드 화합물 Expired - Fee Related KR970010758B1 (ko)

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AUPJ1304/88 1988-11-04
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