A1 Refereed original research article in a scientific journal
RNA interference tolerates 2 '-fluoro modifications at the Argonaute2 cleavage site
Authors: Muhonen P, Tennila T, Azhayeva E, Parthasarathy RN, Janckila AJ, Vaananen HK, Azhayev A, Laitala-Leinonen T
Publisher: WILEY-V C H VERLAG GMBH
Publication year: 2007
Journal: Chemistry and Biodiversity
Journal name in source: CHEMISTRY & BIODIVERSITY
Journal acronym: CHEM BIODIVERS
Volume: 4
Issue: 5
First page : 858
Last page: 873
Number of pages: 16
ISSN: 1612-1872
DOI: https://doi.org/10.1002/cbdv.200790073
Abstract
Short interfering RNA (siRNA) molecules with good gene-silencing properties are needed for drug development based on RNA interference (RNAi). An initial step in RNAi is the activation of the RNA-induced silencing complex RISC, which requires degradation of the sense strand of the siRNA duplex. Although various chemical modifications have been introduced to the antisense strand, modifications to the Argonaute2 (Ago2) cleavage site in the sense strand have, so far, not been described in detail. In this work, novel 2 '-F-purine modifications were introduced to siRNAs, and their biological efficacies were tested in cells stably expressing human tartrate-resistant acid phosphatase (TRACP). A validated siRNA that contains both purine and pyrimidine nucleotides at the putative Ago2 cleavage site was chemically modified to contain all possible combinations of 2 '-fluorinated 2 '-deoxypurines and/or 2 '-deoxypyrimidines in the antisense and/or sense strands. The capacity of T-F-modified siRNAs to knock down their target mRNA and protein was studied, together with monitoring siRNA toxicity. All 2 '-Fmodified siRNAs resulted in target knockdown at nanomolar concentrations, despite their high thermal stability. These experiments provide the first evidence that RISC activation not only allows 2 '-F modifications at the sense-strand cleavage site, but also increase the biological efficacy of modified siRNAs in vitro.
Short interfering RNA (siRNA) molecules with good gene-silencing properties are needed for drug development based on RNA interference (RNAi). An initial step in RNAi is the activation of the RNA-induced silencing complex RISC, which requires degradation of the sense strand of the siRNA duplex. Although various chemical modifications have been introduced to the antisense strand, modifications to the Argonaute2 (Ago2) cleavage site in the sense strand have, so far, not been described in detail. In this work, novel 2 '-F-purine modifications were introduced to siRNAs, and their biological efficacies were tested in cells stably expressing human tartrate-resistant acid phosphatase (TRACP). A validated siRNA that contains both purine and pyrimidine nucleotides at the putative Ago2 cleavage site was chemically modified to contain all possible combinations of 2 '-fluorinated 2 '-deoxypurines and/or 2 '-deoxypyrimidines in the antisense and/or sense strands. The capacity of T-F-modified siRNAs to knock down their target mRNA and protein was studied, together with monitoring siRNA toxicity. All 2 '-Fmodified siRNAs resulted in target knockdown at nanomolar concentrations, despite their high thermal stability. These experiments provide the first evidence that RISC activation not only allows 2 '-F modifications at the sense-strand cleavage site, but also increase the biological efficacy of modified siRNAs in vitro.
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