Thanks to decades of work, we have a great deal of detailed knowledge about how various chemical modifications of a PS ASO can affect hybridization to target RNAs and how those modifications affect the interactions of the heteroduplex with RNAseH1, including binding, catalytic rate and sites of cleavage of the target RNA. However, it is now clear that cellular RNAs may be modified post-transcriptionally with numerous chemical modifications and, to date, very little is known about how post-transcriptional modifications of RNA affect PS ASO binding and RNAse H1 enzymology.
The manuscript by Lacy (Lacy, K.A. et. al., Molecular Therapy Nucleic Acids, 28, 814-828, 2022) represents the first of a series of publications in which we assess the impacts of various RNA modifications on heteroduplex stability and RNAse H1 enzymology. We chose to study three relatively common RNA modifications, 2’methoxy, Inosine (A to I editing) and m6A. The process used included cell-free assessments of each of these modifications made at different positions in cognate RNA target affected hybridization and RNAse H1 enzymology and then we assessed the impact of those modifications on the activity of gapmer PS ASOs designed to serve as substrates for RNAse H1 when in the PS ASO-RNA heteroduplex. The effects of each modification on hybridization were, in the main, as expected. Single 2’ methoxy modifications had little effect on the thermal stability of the heteroduplex, but altered the sites of cleavage in the target RNA induced by RNAse H1 by blocking cleavage at the site of the modification and shifting the cleavage pattern which are consistent with the catalytic mechanism of the enzyme. The changes in cleavage pattern induced by 2’methoxy substation in the RNA differed from the effects observed when placed in the ASO, again consistent with the catalytic mechanism. Further the extent and rate of RNA cleavage by the enzyme were reduced and the effects on these parameters varied as a function of sequences (the number of cleavage sites and cleavage at each site is affected by sequence) and the site in the RNA modified, again consistent with our understanding of the enzyme. However, when the effects of the same modification on PS ASO activity were minor in cells. Many factors may account for less impact on cellular activity of PS ASOs. However, in cells, there may be cluster of 2’methoxys as well as other modifications. Therefore, for ASO design, we recommend avoiding sites known to be ‘methoxy modified.
Inosine creates a mis-match in the heteroduplex. RNAse H1 has been shown to be able to tolerate some mis-matches induced by altering the sequence of the ASO, but as a general rule, mis-matches reduce the thermal stability of the heteroduplex and RNAseH1 cleavage rate and extent. We found that Inosine placed in the RNA also altered the duplex stability and reduced RNAse H1 activity in a manner similar to the effects of Inosine incorporated into the ASO, consistent with expectations. Once again, however, in cells the effects were modest. Nevertheless, in ASO design recommend avoiding sites known to be prone to A to I editing.
Finally, m6A substitution had a very minimal effect on ASO-RNA hybridization and no effect on RNAse H1 binding or cleavage activity, again consistent with expectations. However, m6A is thought to alter interactions with some proteins, including M6A “readers”. Consequently, sites with m6A modifications should be approached with these observations in mind when selecting an ASO.
It is known that cellular RNAs are extensively modified with many other types of modifications. However, the most highly modified cellular RNAs are tRNAs, Pre- and ribosomal RNAs and some small RNAs such as sno-RNAs. In pre- and m-RNAs numerous modifications have been identified, but it is unclear how many of these modifications are reproducibly identifiable and what if any effects on RNA structure, stability or protein interactions they may have. In this study we showed that two modifications, 2’methoxy and Inosine induce pronounced effects on RNAse H1 enzymology in purified systems, but limited effects in cell-based assays. Thus, it is important to develop a catalog of RNA modifications and define their effects on duplex stability and RNAse H1 enzymology and to continue to evaluate the effects in cells on ASO activity in a systematic manner. Moreover, it will be important to examine the effects of clusters of individual modifications and the effects of several types of modifications as well.