Characterization of Escherichia coli RNase H Discrimination of DNA Phosphorothioate Stereoisomers

Abstract

Phosphorothioate (PS) modification of antisense oligonucleotides (ASOs) is a critical factor enabling their therapeutic use. Standard chemical synthesis incorporates this group in a stereorandom manner; however, significant effort was made over the years to establish and characterize the impact of chiral control. In this work, we present our in-depth characterization of interactions between Escherichia coli RNase H and RNA-DNA heteroduplexes carrying chirally defined PS groups. First, using a massive parallel assay, we showed that at least a single R p-PS group is necessary for efficient RNase H-mediated cleavage. We followed by demonstrating that this group needs to be aligned to the phosphate-binding pocket of RNase H, and that chiral status of other PS groups in close proximity to RNase H does not affect cleavage efficiency. We have shown that RNase H's PS chiral preference can be utilized to guide cleavage to a specific chemical bond. Finally, we present a strategy for ASO optimization by mapping preferred RNase H cleavage sites of a non-thioated compound, followed by introduction of R p-PS in a strategic position. This results in a cleaner cleavage profile and higher knockdown activity compared with a compound carrying an S p-PS at the same location.

Discovery and characterization of stereodefined PMO-gapmers targeting tau

“…Thus, single R p walk and SSR walk in the DNA gap region were conducted to find the stereoisomers showing the best activity. Similar strategies have been reported for the identification of the optimal stereochemistry (42, 43). After evaluating the activity of all the synthesized stereoisomers, it was found that R p at specific positions (e.g., at G7-A8 for ASO-409 (Figure 5) and at G8-A9 for ASO-486 (Figure 6)) is crucial to have good knockdown activity.…”

“…In addition, the presence of a second R p linkage did not give significant activity gain (e.g., ASO-409-SSR2-M vs. ASO-409-R3-M, and ASO-486-SSR2-M vs. ASO-486-R5-M). It is reported that the position of R p linkage in the gap region guides the RNase H cleavage site and selection of the position of R p linkage reinforcing the cleavage at the native cleavage sites gives a cleaner cleavage profile and higher knockdown activity (42, 43). The RNase H cleavage site analysis showed that ASO-486-SSR2-R and ASO-486-R5-R result in an increased cleavage at the same cleavage site presumably due to the R p linkage at G8-A9 (Supplementary Figure S7).…”

Recent Progress and Challenges in the Development of Antisense Therapies for Myotonic Dystrophy Type 1

“…It has been known for about 50 years now that this modification makes the oligonucleotides resistant to nuclease degradation, likely because of the larger van der Waals radius of the sulfur atom displacing the metal ions at the active sites of nucleases [ 19 , 20 , 21 ]. Furthermore, PS-modified ASOs enable the cleavage of the hybridized RNA strand by RNase H [ 22 , 23 ]. As the ASO is still active following target degradation, this mechanism dramatically enhances its inhibitory activity.…”

Inhomogeneous Diastereomeric Composition of Mongersen Antisense Phosphorothioate Oligonucleotide Preparations and Related Pharmacological Activity Impairment

“…The replacement of phosphodiester (PO) with phosphorothioate (PS) linkages was the first chemical modification to be investigated and it is still the most extensively used, in particular, in RNase H-dependent ASOs [ 1 , 4 , 6 , 7 ]. In these ASOs, replacement of the PO with PS improves resistance to nucleases and facilitates cellular uptake and bioavailability in vivo without significantly impairing hybridization with the target RNA or the posthybridization RNase H activation [ 1 , 4 , 6 , 8 ].…”