Effects of local mRNA structure on posttranscriptional gene silencing

Abstract

Antisense oligodeoxynucleotides (AONs) and short interfering RNAs (siRNAs) effect posttranscriptional gene silencing (PTGS) by hybridizing to an mRNA and then directing its cleavage. To understand the constraints that mRNA structure imposes on AON-vs. siRNA-mediated PTGS, AON-and siRNA-mediated cleavage of defined mRNA structures was monitored in Drosophila embryo whole-cell lysates. We observed that AON-directed cleavage was Ϸ3-fold faster than cleavage with a siRNA directed to the same target site. Furthermore, and unexpectedly, AON-mediated cleavage was found to be much less fastidious with respect to target sequence accessibility, as measured by the presence of unpaired nucleotides, than a corresponding siRNA. Nonetheless, in vivo, siRNAs silenced their mRNA target at least 2-fold more efficiently than the corresponding AON. These seemingly contradictory results suggested that additional, as yet undefined factors play an important role in regulating PTGS efficiency in vivo. We used a well defined RNA-binding protein, ␣CP, and its corresponding highaffinity RNA-binding site to explore this hypothesis. We found that prebound ␣CP effectively blocked AON-mediated cleavage of the RNA-binding site compared with cleavage of the site in the absence of ␣CP. We conclude that higher-order structures formed by RNA and bound proteins play an important role in determining the efficiency of AON-directed PTGS. We hypothesize that strategies aimed at removing RNA-binding proteins might significantly improve AON-mediated PTGS in vivo.antisense ͉ oligodeoxynucleotide ͉ siRNA

RNA Targeting Therapeutics: Molecular Mechanisms of Antisense Oligonucleotides as a Therapeutic Platform

“…For example, a comparison of a series of optimized RNase H oligonucleotides to siRNA oligonucleotides in cultured cells revealed that they have similar potencies, maximal efficacies, and sequence specificities (30). Others have reported that siRNA oligonucleotides were more potent in cell culture than RNase H oligonucleotides (101,102). In these instances, the RNase H oligonucleotides used were not optimally designed, incorporating low-affinity phosphorothioate-modified oligodeoxynucleotides (first-generation modification) and/or designs in which the oligonucleotide is not stable to nuclease degradation in cell culture.…”

Fluorescent Probes for Live-Cell RNA Detection

“…Further, RNA molecules form complex secondary (folded) structures (Figure 7). Therefore, when designing an oligonucleotide probe, it is preferable to avoid targeting RNA sequences that are double stranded, or occupied by RNA-binding proteins; otherwise, the probe has to compete off the RNA strand or the RNA-binding protein in order to hybridize to the target (63). Indeed, molecular beacons designed for targeting a specific RNA often show no signal when delivered to living cells (62).…”

Silencing disease genes in the laboratory and the clinic

“…Various groups have proposed computational [97–99] or experimental [99–104] methods for identifying potent ASOs. Aspects of mRNA secondary structure can be roughly predicted, but algorithms such as the popular mFOLD do not take into account factors like tertiary structure or the involvement of RNA binding proteins, and so they are of limited utility in practice [105]. Ultimately if a group is serious about finding a potent ASO, they should test as many oligonucleotides as time and budget permit [106,107].…”

“…Several studies have directly compared the activity of various ASOs and siRNAs [105,153–157]. These studies can be misleading if one of the compounds contains suboptimal chemistry or sequence selection (e.g.…”