Abstract
RNA interference (RNAi) is an evolutionary conserved post-transcriptional gene silencing mechanism, in which double stranded RNA effector molecules trigger the degradation of complementary mRNA transcripts. The use of RNAi to reduce gene expression with high specificity and ready availability is a powerful tool for reverse genetics and provides great therapeutic potential for targeting diseases caused by the expression of a deleterious gene or mutant allele, e.g. cancer and viral infections. Besides the known preferences of the RNAi technique, there is a need for the development of improved small double stranded silencing triggers with long lasting silencing activity and maximum specificity. The introduction of chemically modified nucleotides into short interfering RNAs (siRNAs) is currently the method of choice. In this review, we summarize the effects of various modifications on siRNA sub-cellular localization and silencing activity, discuss ideal chemical modifications and positions within siRNAs suited for their use in medical therapies and present a new perspective to study siRNA mediated silencing in vivo by fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) to further improve RNAi-based pharmaceuticals.
Keywords: siRNA, RISC, Argonaute 2, Exportin-5, fluorescence, microinjection, live cell imaging, in vivo FCS/ FCCS
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