利用锁核酸化学偶联FokⅠ核酸酶靶向切割HBV基因的体外实验

doi:10.16288/j.yczz.15-435

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HEREDITAS(Beijing) ›› 2016, Vol. 38 ›› Issue (4): 350-359.doi: 10.16288/j.yczz.15-435

• Research Articles • Previous Articles Next Articles

Locked nucleic acid couples with FokⅠnucleases to target and cleave hepatitis B virus’s gene in vitro

Li Ma¹, Hongyan Chen¹, Huaxing Zhu², Wei Li³, Daru Lu¹

1. State Key Laboratory of Genetic Engineering, School of Life Science, Fudan University, Shanghai 200433, China;
2. Shanghai Novoprotein Technology Co., Ltd., Shanghai 201203, China;
3. Sangon Biotech (Shanghai) Co., Ltd., Shanghai 201611, China

Received:2015-10-17 Revised:2015-11-19 Online:2016-04-20 Published:2016-04-20

Abstract

Abstract: Hepatitis B virus (HBV) is a dented double-stranded DNA virus. After infecting human hepatic cells, it forms cccDNA that replicates persistently and integrates randomly into the host’s genome during the process of reserve transcription. On average, in each cell with chronic HBV infection, there are about 33 copies of cccDNA with a half of 35-57 days, which can be difficult to eradicate. A new strategy is to inhibit HBV transcription by using locked nucleic acid (LNA). Besides, cleaving HBV genome by targeted genome editing technologies could potentially cure patients. In this study, we explored new genome editing tools for HBV treatment. Based on LNA’s ability to form triple helix by binding to duplex DNA, its stability towards nuclease and polymerase, and its sensitivity to single base mismatches, we designed LNA-modified oligonucleotides as DNA binding domain to effectively increase the specificity of target gene recognition. Meanwhile, by utilizing the small molecular weight and dimerization dependent activity of nuclease FokⅠ, we used FokⅠrecombinant dimer protein as DNA cleavage domain. Here, we established a method by chemical coupling of LNA-oligonucleotide with FokⅠcleavage domain, and also validated the targeted cleavage of HBV genes with our new tools in vitro. These results provide new possibilities for future in vivo anti-virus gene therapy with high specificity and no integration risk.

Key words: hepatitis B virus, gene editing, locked nucleic acid, FokⅠ, chemical coupling

Li Ma, Hongyan Chen, Huaxing Zhu, Wei Li, Daru Lu. Locked nucleic acid couples with FokⅠnucleases to target and cleave hepatitis B virus’s gene in vitro[J]. HEREDITAS(Beijing), 2016, 38(4): 350-359.

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Locked nucleic acid couples with FokⅠnucleases to target and cleave hepatitis B virus’s gene in vitro

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