title: Gene silencing activity of siRNAs with a ribo-difluorotoluyl nucleotide.
abstract: Recently, chemically synthesized short interfering RNA (siRNA) duplexes have been used with success for gene silencing. Chemical modification is desired for therapeutic applications to improve biostability and pharmacokinetic properties; chemical modification may also provide insight into the mechanism of silencing. siRNA duplexes containing the 2,4-difluorotoluyl ribonucleoside (rF) were synthesized to evaluate the effect of noncanonical nucleoside mimetics on RNA interference. 5'-Modification of the guide strand with rF did not alter silencing relative to unmodified control. Internal uridine to rF substitutions were well-tolerated. Thermal melting analysis showed that the base pair between rF and adenosine (A) was destabilizing relative to a uridine-adenosine pair, although it was slightly less destabilizing than other mismatches. The crystal structure of a duplex containing rFoA pairs showed local structural variations relative to a canonical RNA helix. As the fluorine atoms cannot act as hydrogen bond acceptors and are more hydrophobic than uridine, there was an absence of a well-ordered water structure around the rF residues in both grooves. siRNAs with the rF modification effectively silenced gene expression and offered improved nuclease resistance in serum; therefore, evaluation of this modification in therapeutic siRNAs is warranted.
authors.affiliation: Alnylam Pharmaceuticals, Inc., 300 Third Street, Cambridge, Massachusetts 02142, USA.
authors.forename: Jie
authors.initials: J
authors.lastname: Xia
authors.forename: Anne
authors.initials: A
authors.lastname: Noronha
authors.forename: Ivanka
authors.initials: I
authors.lastname: Toudjarska
authors.forename: Feng
authors.initials: F
authors.lastname: Li
authors.forename: Akin
authors.initials: A
authors.lastname: Akinc
authors.forename: Ravi
authors.initials: R
authors.lastname: Braich
authors.forename: Maria
authors.initials: M
authors.lastname: Frank-Kamenetsky
authors.forename: Kallanthottathil G
authors.initials: KG
authors.lastname: Rajeev
authors.forename: Martin
authors.initials: M
authors.lastname: Egli
authors.forename: Muthiah
authors.initials: M
authors.lastname: Manoharan
mesh_terms: D020029:Base Pairing; D001483:Base Sequence; D004355:Drug Stability; D020868:Gene Silencing / Q000187:drug effects; D006860:Hydrogen Bonding; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D034741:RNA, Small Interfering / Q000737:chemistry / Q000235:genetics*; D012265:Ribonucleotides / Q000494:pharmacology*
chemical_list: D034741:RNA, Small Interfering; D012265:Ribonucleotides