Fig. 1
Genome-wide scanning of conservative and efficient anti-HBV siRNA triggers. a The structure of HBV RNAs and the location of siRNA candidates. The siRNA candidates are shown as short lines. The locations of four HBV ORFs (HBs, HBc, HBx and Pol) and the relative transcripts (3.5 kb, 2.4 kb, 2.1 kb, and 0.7 kb) are indicated. The distribution of 22 conserved motifs in HBV genome are also indicated. b Twenty siRNA candidates were transfected by RNAiMAX transfection reagents and screened at the dose of 50 nM in HepG2-HBVEGFP cells (HBsAg derived from recombination cccDNA. The reporting cell line was constructed in-house) and PLC/PRF/5 cells (HBsAg derived from HBV integrates. The cell line carrying natural HBV integrates was derived from clinical patents). The supernatants were collected at 72 h post-transfection, and the HBsAg levels in the culture medium were determined by commercial ELISA kits and was normalized to that of cells treated with a negative control siRNA (siNC). HepG2-NTCP (NTCP receptor-overexpressing HepG2 cell line that supports HBV infection) cells were infected with HBV at a multiplicity of infection (MOI) of 200 for 3 days, then transfected with siRNA candidates at the dose of 50 nM using transfection reagents (RNAiMAX). HBeAg expression in the culture medium was analyzed at 72 h post-transfection, and the intracellular pgRNA and total RNA levels were detected with the corresponding primers. c The inhibitory efficacy of siRNAs on EGFP expression levels in HepG2-HBVEGFP cells were visualized by high content imaging (HCI) and screening (HCS) assay using Operetta CLS (PerkinElmer). Scale bar indicated 200 μm. d HepG2-NTCP cells were infected with HBV at the MOI of 200, then treated with serial dilutions of siRNA (0, 6.25, 12.5, 25, 50, and 100 nM) using RNAiMAX transfection reagent. HBeAg expression levels in the supernatants were determined by ELISA kits, and the IC50 values were calculated. The si74 and si77 (two siRNA triggers from ARC-520) were used as positive controls. Data were shown as means ± SDs (n = 3 ~ 4)
