One of the long-term goals of my laboratory has been to understand the cellular factors that influence the activities of antisense oligonucleotides (ASOs). For example, we have used a well-defined and structurally characterized model system, the SOD1 mini-gene, to evaluate the effects of RNA structure, RNA binding proteins, repetitive sequences, the strength of splicing signals, the length and position of introns and other factors on ASO activities. We have used the same system to characterize the kinetics of each step in the process that recruits ribonuclease (RNase) H1 to ASO-RNA duplexes and cleaves the target RNA.
In a paper published in Nucleic Acids Research we asked if the rate of translation might affect the activities of ASOs. We learned several important lessons. First, translation rate may affect the activities of ASOs. For ASOs that bind in the 5’UTR region, translation rate had little effect. For ASOs designed to target the coding region, rapidly translated RNAs were more resistant to ASO-induced RNase H1 mediated degradation than less effectively translated mRNAs. We then demonstrated for the first time that ASOs are active in the polysome and that the main reason that rapid translation reduces ASO activities is competition between RNase H1 and ribosomes that impede RNase H1 from binding to the ASO-RNA duplex. Further, we showed that for ASOs designed to bind in the 3’UTR of the mRNA, reduced translation reduced ASO activity, probably because in the absence of effective translation, the 3’UTR is less accessible for ASO binding.
To fully appreciate the importance of this paper, one must consider it in the context of all the work that we’ve done to define factors influencing ASO activities. This work adds to the catalog of knowledge that helps us understand why many ASOs work while others do not and how the various activities of the cell affect ASO behavior. When we combine this knowledge with growing knowledge about mechanisms of ASO action, we can incorporate all the information into algorithms that enable more efficient design of therapeutic ASOs.