Applied Biological Chemistry

The Korean Society for Applied Biological Chemistry (한국응용생명화학회)
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Construction of a Hammerhead Ribozyme that Cleaves Rice Black-Streaked Dwarf Virus RNA

흑조위축병 바이러스 RNA를 절단하는 망치머리형 라이보자임의 제작

  • Kim, Ju-Kon (Department of Genetic Engineering, Suwon University) ;
  • Sohn, Seong-Han (Biochemistry Division, National Agricultural Science and Technology Institute, RDA) ;
  • Lee, Sug-Soon (Department of Agronomy, Yeungnam University) ;
  • Hwang, Young-Soo (Biochemistry Division, National Agricultural Science and Technology Institute, RDA) ;
  • Park, Jong-Sug (Biochemistry Division, National Agricultural Science and Technology Institute, RDA)
  • 김주곤 (수원대학교 유전공학과) ;
  • 손성한 (농촌진흥청 농업과학기술원 생화학과) ;
  • 이석순 (영남대학교 농학과) ;
  • 황영수 (농촌진흥청 농업과학기술원 생화학과) ;
  • 박종석 (농촌진흥청 농업과학기술원 생화학과)
  • Published : 1995.12.31
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Abstract

To develop an antiviral agent for the rice black-streaked dwarf virus (RBSDV), a hammerhead type ribozyme, which has a potential target site on the genome segment 3, was designed. Oligonucleotides for the ribozyme and its substrate were synthesized, annealed, and cloned into a plasmid pBluescript II KS(+). Ribozyme and substrate RNAs were then synthesized by in vitro transcription with $T_3$ RNA polymerase, obtaining RNAs in expected size, 193 and 182 nucleotides, respectively. The substrate RNA was efficiently cleaved into two fragments when incubated with the ribozyme at $55^{\circ}C$, while the cleavage was not detected at $37^{\circ}C$. In addition, the segment 3 RNA of RBSDV was also cleaved into two fragments by the same ribozyme at $55^{\circ}C$. Taken together, our results demonstrated that the hammerhead ribozyme has an in vitro endonucleolytic activity and may be used as an antiviral agent in transgenic plants.

흑조위축병 바이러스(RBSDV)에 대한 antiviral agent를 개발하기위하여 RBSDV 게놈 조각 3번을 절단하는 망치머리형 구조의 라이보자임을 설계하였다. 라이보자임과 기질의 oligonucleotides는 DNA 합성기로 합성 후 서로 합치고, pBluescript KS(+)에 삽입하였다. 라이보자임과 기질 RNA는 $T_3$ RNA polymerase로 기내 전사하여 각각 193과 183 nucleotide의 RNA를 얻었다. 기질 RNA는 라이보자임 RNA에 의해 $55^{\circ}C$에서 2개의 조각으로 절단되었으나 $37^{\circ}C$에서는 절단되지 않았다. 또한 RBSDV의 3번 조각 RNA도 동일한 라이보자임에 의해 절단되었다. 이러한 결과로 합성된 헤머해드 라이보자임은 기내 절단 능력을 가지며 형질전환 식물체에서 antiviral agent로 이용될 수 있을 것이다.

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