Characterization of Escherichia coli RNase H Discrimination of DNA Phosphorothioate Stereoisomers.
In: Nucleic Acid Therapeutics, Jg. 31 (2021-12-01), Heft 6, S. 383-391
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Zugriff:
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 Rp-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 Rp-PS in a strategic position. This results in a cleaner cleavage profile and higher knockdown activity compared with a compound carrying an Sp-PS at the same location. [ABSTRACT FROM AUTHOR]
Titel: |
Characterization of Escherichia coli RNase H Discrimination of DNA Phosphorothioate Stereoisomers.
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Autor/in / Beteiligte Person: | Kiełpiński, Łukasz J. ; Funder, Erik Daa ; Schmidt, Steffen ; Hagedorn, Peter H. |
Zeitschrift: | Nucleic Acid Therapeutics, Jg. 31 (2021-12-01), Heft 6, S. 383-391 |
Veröffentlichung: | 2021 |
Medientyp: | academicJournal |
ISSN: | 2159-3337 (print) |
DOI: | 10.1089/nat.2021.0055 |
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