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Virologica Sinica, 32 (4) : 331, 2017
Letter
A practical approach to generate suitable de novo synthesis RNA template for a flavivirus RNA-dependent RNA polymerase
1. Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China
2. University of Chinese Academy of Sciences, Beijing 100049, China
 Correspondence: gongpeng@wh.iov.cn
(5432.24KB)  
Abstract
Taken together,we have successfully utilized the HDV ribozyme in the preparation of homogeneous RNA sample that may be free of sequence limitation at the 3'-end.To make the HDV approach-derived RNA suitable for directing de novo RNA synthesis by the WNV NS5 RdRP, the 2',3'-cyclic phosphate at its 3'-end needs to be removed. The cyclic phosphate interference with the WNV RdRP initiation is an interesting observation and the underlying mechanisms may deserve further investigation. The practical approach combining T7 RNAP transcription, HDV ribozyme self-cleavage,and T4 PNK 2',3'-cyclic phosphate removal utilized in this study is likely applicable for in vitro characterization of de novo viral RdRPs.In a broader context,this approach may be applicable for developing RNA-based assays and for X-ray crystallography-based or cryo-electron microscopy (EM)-based structural studies of RNA and RNA-protein complexes.
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Received: 20 Jun 2017  Accepted: 20 Jun 2017  Published online: 20 Jun 2017
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