title: 5'-C-Malonyl RNA: Small Interfering RNAs Modified with 5'-Monophosphate Bioisostere Demonstrate Gene Silencing Activity.
abstract: 5'-Phosphorylation is a critical step in the cascade of events that leads to loading of small interfering RNAs (siRNAs) into the RNA-induced silencing complex (RISC) to elicit gene silencing. 5'-Phosphorylation of exogenous siRNAs is generally accomplished by a cytosolic Clp1 kinase, and in most cases, the presence of a 5'-monophosphate on synthetic siRNAs is not a prerequisite for activity. Chemically introduced, metabolically stable 5'-phosphate mimics can lead to higher metabolic stability, increased RISC loading, and higher gene silencing activities of chemically modified siRNAs. In this study, we report the synthesis of 5'-C-malonyl RNA, a 5'-monophosphate bioisostere. A 5'-C-malonyl-modified nucleotide was incorporated at the 5'-terminus of chemically modified RNA oligonucleotides using solid-phase synthesis. In vitro silencing activity, in vitro metabolic stability, and in vitro RISC loading of 5'-C-malonyl siRNA was compared to corresponding 5'-phosphorylated and 5'-nonphosphorylated siRNAs. The 5'-C-malonyl siRNAs showed sustained or improved in vitro gene silencing and high levels of Ago2 loading and conferred dramatically improved metabolic stability to the antisense strand of the siRNA duplexes. In silico modeling studies indicate a favorable fit of the 5'-C-malonyl group within the 5'-phosphate binding pocket of human Ago2MID domain.
authors.affiliation: Alnylam Pharmaceuticals , 300 Third Street, Cambridge, Massachusetts 02142, United States.
authors.forename: Ivan
authors.initials: I
authors.lastname: Zlatev
authors.affiliation: Alnylam Pharmaceuticals , 300 Third Street, Cambridge, Massachusetts 02142, United States.
authors.forename: Donald J
authors.initials: DJ
authors.lastname: Foster
authors.affiliation: Alnylam Pharmaceuticals , 300 Third Street, Cambridge, Massachusetts 02142, United States.
authors.forename: Jingxuan
authors.initials: J
authors.lastname: Liu
authors.affiliation: Alnylam Pharmaceuticals , 300 Third Street, Cambridge, Massachusetts 02142, United States.
authors.forename: Klaus
authors.initials: K
authors.lastname: Charisse
authors.affiliation: Alnylam Pharmaceuticals , 300 Third Street, Cambridge, Massachusetts 02142, United States.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Brigham
authors.affiliation: Alnylam Pharmaceuticals , 300 Third Street, Cambridge, Massachusetts 02142, United States.
authors.forename: Rubina G
authors.initials: RG
authors.lastname: Parmar
authors.affiliation: Alnylam Pharmaceuticals , 300 Third Street, Cambridge, Massachusetts 02142, United States.
authors.forename: Vasant
authors.initials: V
authors.lastname: Jadhav
authors.affiliation: Alnylam Pharmaceuticals , 300 Third Street, Cambridge, Massachusetts 02142, United States.
authors.forename: Martin A
authors.initials: MA
authors.lastname: Maier
authors.affiliation: Alnylam Pharmaceuticals , 300 Third Street, Cambridge, Massachusetts 02142, United States.
authors.forename: Kallanthottathil G
authors.initials: KG
authors.lastname: Rajeev
authors.affiliation: Department of Biochemistry, Vanderbilt University , Nashville, Tennessee 37232, United States.
authors.forename: Martin
authors.initials: M
authors.lastname: Egli
authors.affiliation: Alnylam Pharmaceuticals , 300 Third Street, Cambridge, Massachusetts 02142, United States.
authors.forename: Muthiah
authors.initials: M
authors.lastname: Manoharan
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D020868:Gene Silencing; D008314:Malonates / Q000737:chemistry*; D051379:Mice; D010766:Phosphorylation; D034741:RNA, Small Interfering / Q000235:genetics*
chemical_list: D008314:Malonates; D034741:RNA, Small Interfering; C030290:malonic acid