Design, Synthesis And Biological Activity Of Chemical Modified SiRNA

Short interfering RNA (siRNA) molecules have drawn considerable attention since it was demonstrated that they mediate potent gene knock-down in a variety of mammalian cells without triggering non-specific RNA degradation and translation inhibition based on interferon response. Argonaute protein, a key component of RISC, with active endonuclease domains contribute to the maturation of bound short interfering RNAs (siRNAs) by degradative cleavage of the passenger strand and subsequent guide-strand-mediated sequence-specific cleavage of target RNAs. Considering the interaction between Ago protein and siRNA, the optimal modification techniques were utilized according to the crystal structure of Ago protein. in order to establish a novel technology of chemical modified siRNA and obtain the modified MDM2-siRNA with high specificity, activity, and stability.Part 1. Chemical modification sites and biological activityFive different siRNA sequences inhibiting the virus replication were selected to verify the relevance of FANA substituted sites and biological activity. The results revealed that:1. The modification with FANA on 3'end of both sense and antisense strand, could reduce the gene silencing effect in different levels. In particular, the modification on 3' end of antisense strand reduced activity more significantly.2. The modification on both 3'end and middle of antisense strand could reduce siRNA activity. In particular, the modification on 9-11 base in the middle of the antisense strand even more significantly reduced activity.3. The modification on sense strand had a relatively slight effect on gene silencing activity, which was affected in varying degrees by different regions in the middle segment of siRNA sequence. The order of the activity was shown as follows:D district> C district> B district, in which the modification on D district may increase the gene silencing effect to some extent.4. The synthesis of FANA monomers was completed for 5 different target sequences. A total of 44 new chemical modified siRNAs were explored. As a result,â…¡-siRNA6,â…¡-siRNA11 andâ…¤-siRNA3 showed the significant inhibitory activity of WSN virus and deserved further evaluation. Part 2. The Theoretical Study on PAZ domain of the Ago proteinBased on the crystal structure of PAZ domain of Ago proteins, the X, Y and Z Series of chemically modified monomers were designed. The relationship between 3'-terminal modification and biological activity could be predicted and evaluated. The results showed that:1.1SI2 and 1SI3 crystal protein were Superposition perfectly, so that PAZ has a conservative structure. The mode of the interaction of the siRNA and Ago protein was analyzed with 1SI2 as representative. The data showed that the 3'end of siRNA could interact with PAZ pocket area with specific binding. The introduction of appropriate hydrophobic groups would be benefit to their interaction.2. X and Z Series of modification monomers were selected. The trend in affinity energy and the combination of different modified monomers on 3'end was preliminarily predicted and evaluated with MacroModel module. The data showed that modified combination including dTX had the smallest total binding energy and van der Waals forces binding energy.3. Used computer-aided molecule alignment method, the different modification combinations on 3'end and the spatial state of PAZ protein were preliminarily predicted and evaluated. The data showed that the dTX modified bases had the same spatial structures with the unmodified dangling end bases. The pyrimidine structure replaced by hydrophobic group in dTX could contribute to the binding of the hydrophobic aromatic fragment and the surrounding hydrophobic amino acid residues by weak van der Waals electrostatic interaction.4. The total binding energy could be significantly affected by the spatial structure of chemically modified monomers, the fragment of the substituent group, and the electrical negativity of partial binding sites. As an instance, the introduction of the hydrophobic segment can reduce the total binding energy. Meanwhile, dTX modification had the same spatial structure with dTdT modification. So the hydrophobic region of aromatic bases could be more effective to reduce binding energy of the 3'end of siRNA and PAZ domain. Reasonably, dTX modification could be most likely to play a role in enhancing the effect of gene silencing.Part 3. The study on terminal modification of siRNAX, Yand Z series of monomers were designed and synthesized. The activities of the modified siRNAs were screened by luciferase reporter system (PGL-4) on HepG2 cells. The results revealed that:1. Modified siRNAs showed lower cytotoxicity than the unmodified siRNAs, with no significant cytotoxic effect at 500 nM concentration.2. The siRNAs with dTX modified antisense strand and variant sense strand exhibited greater silencing effect.3. The 3'end modification could significantly affect the siRNA's activity, which implied that the specific recognition of PAZ and siRNA plays a critical role in gene silence and the key modification site may be the 3'end bases.4. The modification pattern XdT/dTX and dTX/dTX (sense/antisense) had the relatively higher activity.Part 4. The study on chemically modified MDM2-siRNAThe modified MDM2-siRNAs were synthesized and evaluated for their activity and stability.1. The activity of MDM2 gene silencing could be significantly enhanced when the original siRNA was modified with monomers as XdT/dTX, dTX/dTX and dTX/XX.2. The stability of siRNA could also be improved by these modification compositions, in which siRNA modified with dTX/dTX was the most resistant to RNase H.3. Theâ…¦-siRNA6 was 2-fold,2-fold and 3-fold compared with the unmodified siRNA in silencing bioactivity, stability and in vitro antitumor activity, respectively. The siRNA could be taken as the candidate for further study.The results indicated that the unwinding of siRNA was completed by the bases in C district of antisense strand and B district of sense strand coordinated with Ago protein. Once the FANA modification took place in the middle site, the unwinding of the siRNA could be easily prevented. Similarly, the binding of siRNA and Ago protein as well as unwinding of the siRNA could be significantly affected by the modification at 3' terminal of either siRNA strand, especially the antisense one.Based on the specific binding characteristics of the 3'end of siRNA and the Ago protein, the theoretical calculation and biological activity screening were applied to preliminarily confirmed that the binding energy theoretically was relevant to the biological activity. The relationship between modification pattern and activity could be predicted by the preliminary evaluation of the binding energy. In addition, the modification pattern obtained by the theoretical data could significantly improve the gene silencing effect of different target sequences, which may implied a general application. In particularity, the dTX/dTX modification pattern could significantly enhance the MDM2-siRNA gene silencing effect, stability, and anti-tumor activity in vitro.