title: Versatile site-specific conjugation of small molecules to siRNA using click chemistry.
abstract: We have previously demonstrated that conjugation of small molecule ligands to small interfering RNAs (siRNAs) and anti-microRNAs results in functional siRNAs and antagomirs in vivo. Here we report on the development of an efficient chemical strategy to make oligoribonucleotide-ligand conjugates using the copper-catalyzed azide-alkyne cycloaddition (CuAAC) or click reaction. Three click reaction approaches were evaluated for their feasibility and suitability for high-throughput synthesis: the CuAAC reaction at the monomer level prior to oligonucleotide synthesis, the solution-phase postsynthetic "click conjugation", and the "click conjugation" on an immobilized and completely protected alkyne-oligonucleotide scaffold. Nucleosides bearing 5'-alkyne moieties were used for conjugation to the 5'-end of the oligonucleotide. Previously described 2'- and 3'-O-propargylated nucleosides were prepared to introduce the alkyne moiety to the 3' and 5' termini and to the internal positions of the scaffold. Azido-functionalized ligands bearing lipophilic long chain alkyls, cholesterol, oligoamine, and carbohydrate were utilized to study the effect of physicochemical characteristics of the incoming azide on click conjugation to the alkyne-oligonucleotide scaffold in solution and on immobilized solid support. We found that microwave-assisted click conjugation of azido-functionalized ligands to a fully protected solid-support bound alkyne-oligonucleotide prior to deprotection was the most efficient "click conjugation" strategy for site-specific, high-throughput oligonucleotide conjugate synthesis tested. The siRNA conjugates synthesized using this approach effectively silenced expression of a luciferase gene in a stably transformed HeLa cell line.
authors.affiliation: Drug Discovery, Alnylam Pharmaceuticals, Cambridge, Massachusetts 02142, USA.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Yamada
authors.forename: Chang Geng
authors.initials: CG
authors.lastname: Peng
authors.forename: Shigeo
authors.initials: S
authors.lastname: Matsuda
authors.forename: Haripriya
authors.initials: H
authors.lastname: Addepalli
authors.forename: K Narayanannair
authors.initials: KN
authors.lastname: Jayaprakash
authors.forename: Md Rowshon
authors.initials: MR
authors.lastname: Alam
authors.forename: Kathy
authors.initials: K
authors.lastname: Mills
authors.forename: Martin A
authors.initials: MA
authors.lastname: Maier
authors.forename: Klaus
authors.initials: K
authors.lastname: Charisse
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Sekine
authors.forename: Muthiah
authors.initials: M
authors.lastname: Manoharan
authors.forename: Kallanthottathil G
authors.initials: KG
authors.lastname: Rajeev
mesh_terms: D000480:Alkynes / Q000737:chemistry*; D001386:Azides / Q000737:chemistry*; D002384:Catalysis; D057930:Click Chemistry; D003300:Copper / Q000737:chemistry; D003500:Cyclization; D015394:Molecular Structure; D009705:Nucleosides / Q000737:chemistry*; D034741:RNA, Small Interfering / Q000737:chemistry*; D013237:Stereoisomerism
chemical_list: D000480:Alkynes; D001386:Azides; D009705:Nucleosides; D034741:RNA, Small Interfering; D003300:Copper