Biological and physicochemical characterization of siRNAs modified with 2′,2′-difluoro-2′-deoxycytidine (gemcitabine)

The use of synthetic short interfering RNAs (siRNAs) is currently a method of choice to manipulate gene expression in mammalian cells. Efforts aimed at improving siRNA biological activity, including increased silencing properties, higher substrate specificity and cellular stability, lower cytotoxici...

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Bibliographic Details
Published in:New journal of chemistry 2010, Vol.34 (5), p.918-924
Main Authors: Sierant, Malgorzata, Sobczak, Milena, Janicka, Magdalena, Paduszynska, Alina, Piotrzkowska, Danuta
Format: Article
Language:English
Subjects:
Antisense
C.D
Chemical modification
Cytotoxicity
gemcitabine
Gene silencing
Mammalian cells
Melting
Seeds
siRNA
Substrate specificity
Thermal stability
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Summary:The use of synthetic short interfering RNAs (siRNAs) is currently a method of choice to manipulate gene expression in mammalian cells. Efforts aimed at improving siRNA biological activity, including increased silencing properties, higher substrate specificity and cellular stability, lower cytotoxicity, and improved target delivery, have been made through the introduction of various chemical modifications into the siRNA strands. In these studies, we present the synthesis of oligoribonucleotides with the single replacement of a cytidine unit for 2",2[prime or minute]-difluoro-2"-deoxycytidine (gemcitabine, dFdC) and the use of them in a series of siRNAs for gene silencing experiments. The dFdC modifications are located in six different positions of the antisense strand, which are crucial for siRNA silencing activity. The results indicate a position-dependent tolerance for the dFdC modification. Gemcitabine units present in the "seed region", at positions 1 or 8, resulted in only a [similar]15% silencing activity in the corresponding duplexes. The dFdC unit at position 10 virtually switched off the silencing activity (below 10%), while the dFdC unit at the positions 2, 4 or 5 produced duplexes of silencing potential comparable to that of the non-modified duplex (70% silencing). The dFdC modification had little impact on the structure of the siRNA duplexes, as determined by circular dichroism analysis, while melting experiments showed their lower thermal stability.
ISSN:1144-0546
1369-9261
DOI:10.1039/b9nj00746f