siRNA target site secondary structure predictions using local stable substructures

Abstract

The crystal structure based model of the catalytic center of Ago2 revealed that the siRNA and the mRNA must be able to form an A-helix for correct positing of the scissile phosphate bond for cleavage in RNAi. This suggests that base pairing of the target mRNA with itself, i.e. secondary structure, must be removed before cleavage. Early on in the siRNA design, GC-rich target sites were avoided because of their potential to be involved in strong secondary structure. It is still unclear how important a factor mRNA secondary structure is in RNAi. However, it has been established that a difference in the thermostability of the ends of an siRNA duplex dictate which strand is loaded into the RNA-induced silencing complex. Here, we use a novel secondary structure prediction method and duplex-end differential calculations to investigate the importance of a secondary structure in the siRNA design. We found that the differential duplex-end stabilities alone account for functional prediction of 60% of the 80 siRNA sites examined, and that secondary structure predictions improve the prediction of site efficacy. A total of 80% of the non-functional sites can be eliminated using secondary structure predictions and duplex-end differential.

Problems associated with reporter assays in RNAi studies

“…A basic approach that is often employed is to design several small RNAs targeting different regions of the same transcript to find the most efficacious inhibitors of gene expression. 10,11 Monitoring of target knockdown can be determined by qPCR (RT-PCR) or northern and western blotting or indirectly by using reporter genes fused to the target of interest. Although the RT-PCR, northern and western blotting approaches are more relevant to monitoring the inhibition of the endogenous gene of interest, the reporter assay has the advantage in that it allows a facile assessment of the activities of both strands of an siRNA or miRNA.…”

The effect of temperature on gene silencing by siRNAs: Implications for silencing in the anterior chamber of the eye

“…It is unclear what the exact temperature of the trabecular meshwork might be, but with the aqueous humor being several degrees lower than body temperature, it is likely the trabecular meshwork will also be below 37°C. It has been shown that silencing is influenced by multiple parameters (Chen et al, 2004;Heale et al, 2005;Overhoff et al, 2005;Schubert et al, 2005). While the SSi is not the only factor that might be influenced by decreased temperatures, changes in SSi with temperature led us to check the effectiveness of siRNAs for myocilin when the silencing was done at less than 37°C.…”

“…The level of silencing was increased almost 1.9 fold with this siRNA as a result of a temperature decrease of 4°C. Again, this silencing increase could be, in part, the result of a change in folding of the mRNA that made this region more single-stranded and allowed better association of the antisense of the siRNA in the RISC complex with the mRNA (Heale et al, 2005;Mittal, 2004).…”

“…This technique can also be used to silence mutated genes that might be related to disease states. Gene silencing is significantly influenced by many parameters, one of which is the folding of the mRNA (Chen et al, 2004;Heale et al, 2005;Luo, Chang, 2004;Schubert et al, 2005). The effect of secondary structure can be quantified by obtaining the single-stranded index (SSi) of an mRNA.…”

Exact calculation of loop formation probability identifies folding motifs in RNA secondary structures

“…RNA secondary structure, defined as the set of A-T, C-G, and G-U canonical pairs in an RNA structure, is a resolution that has proven useful for studying RNA. Secondary structures have been used to find functional RNA in genomes (Macke et al 2001;Klein and Eddy 2003;Torarinsson et al 2006;Uzilov et al 2006;Yao et al 2006;Nawrocki et al 2009;Gorodkin et al 2010;Gruber et al 2010;Fu et al 2015), to find regions of an RNA that are accessible for binding by siRNAs (Heale et al 2005;Lu and Mathews 2007;Tafer et al 2008), and for designing RNAs with desired structures or functions (Hofacker et al 1994;Zadeh et al 2010;Garcia-Martin et al 2013;Lee et al 2014). Secondary structure prediction can also be used to search for motifs of interest, such as binding sites for small molecules (Velagapudi et al 2014) or proteins (Re et al 2014).…”