Optimization Of SiRNA Design Targeting Against HBV

RNAi, a fast-growing area, capture the imagination with their promise of rational drug design and exquisite specifity. But the design and selectivity of effective siRNA is still with low efficiency and by random. With the rapid development of human genome project and transcriptome, it challenges greatly the siRNA optimal design considering the complex genome target with high mutation, heterogeneity, and overlapping genes. HBV, the smallest double-strand DNA virus genome infected the human, with four overlapping ORF, high mutation and at least eight genotypes, is the appropriate target for preliminary analysis of siRNA QSAR. In this study, we try to set the HBV mRNA as target and optimize the siRNA design comprehensively basing on the primary and secondary structure affect respects. Several rules were revealed on the optimal siRNA design by QSAR analysis evaluated by quantative methods. Based on the results, the influence brought by endpoints on siRNA was investigated. Several excellent sequences with good activities were further studied their drugable ability.This dissertation consists of two main parts (Four chapters totally):Preface The importance of siRNA optimal design and the necessary of QSAR analysis were briefly described. The existing study of RNAi on HBV and related problem were also introduced, based on all which, the aim and content of the dissertation were made.PartⅠ(ChapterⅠ,ⅡandⅢ) A structure–activity relationship study on the silencing effect of siRNA on HBV S/P mRNA and HBsAg expressionTwenty-two synthetic siRNAs (including positive controls) against HBV mRNA were evaluated, and their target mRNA genotypic homology, secondary structural elements of mRNA and several free energy△G0 parameters were analyzed to investigate their structure–activity relationships. High-throughput quantative methods, HBsAg ELISA and mRNA real-time PCR were developed. The in-vitro inhibitory effect of siRNAs at 1, 10, and 100 nM on the expression of S/P/pregenomic mRNA and HBsAg were determined quantitatively in HepG2.2.15 cells.Results showed that the 21 siRNAs exhibited diverse efficacy, with 15 out of 21 showing significant knockdown efficacy compared with the control group (P < 0.05). Several sequences were better than the positive control (such as 537, 672, 1658 and etc) which can be chosen as candidate sequence for further siRNA drugable study. About half of (13/21) the siRNAs showed significant concentration-dependent silencing effects on both (P < 0.05).Multiple stepwise linear regression analysis based on the primary structures showed that the genotypic homology, ORF X had tight correlation with the efficacy of siRNAs on inhibiting the S/P mRNA expressions (P < 0.001 and P < 0.05). And the inhibition ability of siRNA on HBsAg expression was associated with homology, S/P, and X (R = 0.692, P < 0.001, R = 0.822, P < 0.01 and R = 0.883, P < 0.01). Noticeably, for both primary analysis of mRNA and HBsAg, the presence of the C gene decreased the efficacy of siRNAs (P < 0.05).Up to ten optimal and suboptimal secondary structures of each target mRNA (pregenomic RNA, polymerase mRNA and HBsAg mRNA) were produced by software RNAstructure 4.4. Target secondary structure was further investigated by asking whether there were any structural patterns in the overall orientation of the Watson-Crick pairing within the immediate region of the guide strand of each siRNA, i.e. numbers of nucleotides in the stem, hairpin, bulge loop, internal loop, and the multiple branch loops. Also, the appearance frequency of the same secondary structures of guide strand of siRNA in the overlapping mRNAs was counted. Finally, relationships between secondary structure and efficacy of siRNAs were analyzed. Stepwise regression, weighted by S/P (primary structural factor), analysis revealed that the number of nucleotides in the hairpin of the S/P/pregenomic mRNA target site played a major role in increasing efficacy (R = 0.877, P = 0.009). By contrast, the multi-branch loop and bulge loop were two factors decreasing efficacy, with the R value increasing to 0.969 (P = 0.04) and 0.994 (P = 0.02), respectively. The predicted values of test siRNAs, calculated based on the regression equation, were consistent with the observed ones. The free energy parameters of siRNA seemed to be irrespective with the siRNA efficacy. During the analysis of each siRNA targeted to S/P/pregenomic overlapping regions of different mRNAs, we found that siRNAs bound to the same secondary structures. The frequencies were≥50% among the total 30 lowest or lower free energy putative structures observed. It is possible that one siRNA targets more than two mRNAs and inhibits the expression of multiple mRNAs. This phenomenon implicated that these secondary structures might be the conserved region which was the target for siRNAs, which might be an important finding for optimal siRNA design.PartⅡ(ChapterⅣ) Roles of different determined endpoints in siRNA efficacy evaluationHBV had multiple overlapping genes and several products expressed simultaneously. In the preliminary study,one important phenomenon was that siRNAs targeting the ORF S/P showed much more efficacy than the ones targeting the ORF C in inhibiting the expressions of S/P mRNA and HBsAg. However, other studies had demonstrated that siRNAs targeting certain ORF of HBV could inhibit gene products of other ORFs. However, whether the comprehensive inhibition was equal or selective needed to be investigated further which would be helpful for optimal siRNA design. Therefore, three effective siRNAs reported were added in our further study, and quantitative HBeAg ELISA methods, real-time QPCR of C/P mRNA and X mRNA, HBV DNA load determination and cccDNA load in HepG2.2.15 cells were developed and employed. The inhibition of twenty-four siRNAs at the concentration of 10nM on different determined endpoints was investigated. Stepwise multiple linear regression analysis showed that there's tight relationship between the target ORF and endpoints. Firstly, the ANOVA showed that the compositor of siRNA efficacy was determined by the determined endpoints (S/P mRNA/HBsAg and C mRNA/HBeAg). Secondly, the siRNAs targeting ORF C/P were more effective than ORF S/P in inhibiting the expressions of C/P mRNA and HBeAg (P < 0.05), and vice versa (P < 0.05). In summary, our studies showed that for the complex overlapping genes, different determined endpoints were important for evaluating the siRNA efficacy. However, key point may exist for the inhibition of final product (for example, for HBV, the HBV DNA load is the final product), and the clarification of the key points was important for optimal drug design.