An efficient RNA-cleaving DNA enzyme can specifically target the 5'-untranslated region of severe acute respiratory syndrome associated coronavirus (SARS-CoV).
BACKGROUND
The worldwide epidemic of severe acute respiratory syndrome (SARS) in 2003 was caused by a novel coronavirus called SARS-CoV. We report the use of DNAzyme (catalytic DNA) to target the 5'-untranslated region (5'UTR) of a highly conserved fragment in the SARS genome as an approach to suppression of SARS-CoV replication. A mono-DNA enzyme (Dz-104) possessing the 10-23 catalytic motif was synthesized and tested both in vitro and in cell culture.


METHODS
SARS-CoV total RNA was isolated, extracted from the SARS-CoV-WHU strain and converted into cDNA. We designed a RNA-cleaving 10-23 DNAzyme targeting at the loop region of the 5'UTR of SARS-CoV. The designed DNAzyme, Dz-104, and its mutant version, Dz-104 (mut), as a control consist of 9 + 9 arm sequences with a 10-23 catalytic core. In vitro cleavage was performed using an in vitro transcribed 5'UTR RNA substrate. A vector containing a fused 5'UTR and enhanced green fluorescent protein (eGFP) was co-transfected with the DNAzyme into E6 cells and the cells expressing eGFP were visualized with fluorescence microscopy and analyzed by fluorescence-activated cell sorting (FACS).


CONCLUSIONS
Our results demonstrated that this DNAzyme could efficiently cleave the SARS-CoV RNA substrate in vitro and inhibit the expression of the SARS-CoV 5'UTR-eGFP fusion RNA in mammalian cells. This work presents a model system to rapidly screen effective DNAzymes targeting SARS and provides a basis for potential therapeutic use of DNA enzymes to combat the SARS infection.
Wu
Shuwen
S
Modern Virology Research Center, State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China.
Xu
Junqiang
J
Liu
Jiangxia
J
Yan
Xuan
X
Zhu
Xiaodong
X
Xiao
Gengfu
G
Sun
Lunquan
L
Tien
Po
P
D020121:5' Untranslated Regions; D000818:Animals; D001483:Base Sequence; D002522:Chlorocebus aethiops; D004247:DNA / Q000378:metabolism*; D017931:DNA Primers; D021881:DNA, Catalytic / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012313:RNA / Q000378:metabolism*; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics*; D014709:Vero Cells
D020121:5' Untranslated Regions; D017931:DNA Primers; D021881:DNA, Catalytic; D012313:RNA; D004247:DNA