title: Automated parallel synthesis of 5'-triphosphate oligonucleotides and preparation of chemically modified 5'-triphosphate small interfering RNA.
abstract: A fully automated chemical method for the parallel and high-throughput solid-phase synthesis of 5'-triphosphate and 5'-diphosphate oligonucleotides is described. The desired full-length oligonucleotides were first constructed using standard automated DNA/RNA solid-phase synthesis procedures. Then, on the same column and instrument, efficient implementation of an uninterrupted sequential cycle afforded the corresponding unmodified or chemically modified 5'-triphosphates and 5'-diphosphates. The method was readily translated into a scalable and high-throughput synthesis protocol compatible with the current DNA/RNA synthesizers yielding a large variety of unique 5'-polyphosphorylated oligonucleotides. Using this approach, we accomplished the synthesis of chemically modified 5'-triphosphate oligonucleotides that were annealed to form small-interfering RNAs (ppp-siRNAs), a potentially interesting class of novel RNAi therapeutic tools. The attachment of the 5'-triphosphate group to the passenger strand of a siRNA construct did not induce a significant improvement in the in vitro RNAi-mediated gene silencing activity nor a strong specific in vitro RIG-I activation. The reported method will enable the screening of many chemically modified ppp-siRNAs, resulting in a novel bi-functional RNAi therapeutic platform.
authors.affiliation: Department of Drug Discovery, Alnylam Pharmaceuticals, 300 Third Street, Cambridge, MA 02142, USA. izlatev@ontorii.com
authors.forename: Ivan
authors.initials: I
authors.lastname: Zlatev
authors.forename: Jeremy G
authors.initials: JG
authors.lastname: Lackey
authors.forename: Ligang
authors.initials: L
authors.lastname: Zhang
authors.forename: Amy
authors.initials: A
authors.lastname: Dell
authors.forename: Kathy
authors.initials: K
authors.lastname: McRae
authors.forename: Sarfraz
authors.initials: S
authors.lastname: Shaikh
authors.forename: Richard G
authors.initials: RG
authors.lastname: Duncan
authors.forename: Kallanthottathil G
authors.initials: KG
authors.lastname: Rajeev
authors.forename: Muthiah
authors.initials: M
authors.lastname: Manoharan
mesh_terms: D000818:Animals; D001331:Automation; D002478:Cells, Cultured; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D015394:Molecular Structure; D009841:Oligonucleotides / Q000138:chemical synthesis* / Q000737:chemistry; D011122:Polyphosphates / Q000737:chemistry*; D034741:RNA, Small Interfering / Q000138:chemical synthesis* / Q000737:chemistry
chemical_list: D009841:Oligonucleotides; D011122:Polyphosphates; D034741:RNA, Small Interfering; C005692:triphosphoric acid