Abstract
RNA interference (RNAi) is a highly specialized process of protein-siRNA interaction that results in the regulation of gene expression and cleavage of target mRNA. The PAZ domain of the Argonaute proteins binds to the 3' end of siRNA, and during RNAi the attaching end of the siRNA switches between binding and release from its binding pocket. This biphasic interaction of the 3' end of siRNA with the PAZ domain is essential for RNAi activity; however, it remains unclear whether stronger or weaker binding with PAZ domain will facilitate or hinder the overall RNAi process. Here we report the correlation between the binding of modified siRNA 3' overhang analogues and their in vivo RNAi efficacy. We found that higher RNAi efficacy was associated with the parameters of lower Ki value, lower total intermolecular energy, lower free energy, higher hydrogen bonding, smaller total surface of interaction and fewer van der Waals interactions. Electrostatic interaction was a minor contributor to compounds recognition, underscoring the presence of phosphate groups in the modified analogues. Thus, compounds with lower binding affinity are associated with better gene silencing. Lower binding strength along with the smaller interaction surface, higher hydrogen bonding and fewer van der Waals interactions were among the markers for favorable RNAi activity. Within the measured parameters, the interaction surface, van der Waals interactions and inhibition constant showed a statistically significant correlation with measured RNAi efficacy. The considerations provided in this report will be helpful in the design of new compounds with better gene silencing ability.
Highlights
RNA interference (RNAi) is a cellular process triggered by double stranded RNA(dsRNA) and regulates the gene expression of target mRNA [1,2]
Dicer is involved in cleavage of microRNA into small interfering RNA, whereas Argonaute proteins (Agos) are the catalytic components of RNA-induced silencing complex (RISC) which bind to siRNAs and cleave mRNA targets [3,4]
In our investigation of the forces governing the recognition of siRNA by the PAZ domain and their in vivo association, we found that weaker binding is correlated with higher RNAi
Summary
RNA interference (RNAi) is a cellular process triggered by double stranded RNA(dsRNA) and regulates the gene expression of target mRNA [1,2]. The major players in this process are the Dicer and Argonaute proteins (Agos). Dicer is involved in cleavage of microRNA (miRNA) into small interfering RNA (siRNA), whereas Agos are the catalytic components of RNA-induced silencing complex (RISC) which bind to siRNAs and cleave mRNA targets [3,4]. The RNAs class that binds with Ago protein, the siRNA, is characterized by the presence of two single nucleotides at their 3’ overhangs. Modified 3’ overhang analogues were an interesting target for development of potent RNAi efficacy [10,11,12]
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