Part:BBa_K1180000
anti-HBV siRNA
Introduction
Small interfering RNA (siRNA) is emerging as a promising therapeutic drug against a wide array of diseases. siRNA can function through the RNA interference pathway to destroy disease-related gene. In order to use siRNA to silence the HBV mRNA[1][2], we need to choose the siRNA target sites. Firstly, we should find 21 nt sequences in the target mRNA from Hepatitis b virus (HBV) genome that begin with an AA dinucleotide. And then, choosing target sites from among the ‘AA sequences’ based on guidelines like ‘Target sequence should have a GC content of around 50%’; ‘’Avoid regions with GC content <30% or > 60%.’ We completed the first two steps in the software, siRNA Designer. Finally, we performed BLAST homology search to avoid off-target effects on other genes or sequences After screening the HBV genome using the methods mentioned above, we ultimately find three candidates as our anti-HBV siRNA. Then we experimentally validate the expression of these three siRNA and chose to use siRNA 467 as our killing module for HBV.
Characterization
After transfection with the siRNA 467 plasmid, the relative expression level of siRNA 467 was tested in HEK 293T cells by quantitative PCR. The result suggested that 467 siRNA had significant expression compared to the negative control (Fig.1).
Fig.1 qPCR analysis of relative siRNA level in 293t cells. Result showed that 467 siRNA has a relatively higher level of expression than that of 308 siRNA and 516 siRNA. </br> After we confirmed the expression of siRNA in HEK 293T cells. The silencing effectiveness of siRNA 467 towards HBsAG was further validated. By qPCR analysis of HepG2 cell co-transfected with both siRNA 467 plasmid and HBsAg plasmid, we observed the significant down-regulation of HBsAg gene (Fig.2). This demonstrated that our siRNA 467 does function to silence the target gene.
Fig. 2 Silencing of HBsAg by siRNA 467. All groups were transfected by Lipo 2000. Control group was transfected with empty plasmid. siRNA-free group was transfected with HBsAg overexpression plasmid, and experimental group with both HBsAg overexpression plasmid and 467 siRNA plasmid. The result suggested that 467 siRNA successfully down-regulatethe HBsAg gene.
References
Morrissey D V, Lockridge J A, Shaw L, et al. Potent and persistent in vivo anti-HBV activity of chemically modified siRNAs[J]. Nature biotechnology, 2005, 23(8): 1002-1007.
Hamasaki K, Nakao K, Matsumoto K, et al. Short interfering RNA-directed inhibition of hepatitis B virus replication[J]. FEBS letters, 2003, 543(1): 51-54.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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