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Curr Protoc Nucleic Acid Chem. 2012 Sep;Chapter 1:Unit1.28. doi: 10.1002/0471142700.nc0128s50.

Solid-phase chemical synthesis of 5'-triphosphate DNA, RNA, and chemically modified oligonucleotides.

Author information

1
Department of Drug Discovery, Alnylam Pharmaceuticals, Cambridge, Massachusetts, USA.

Abstract

A chemical method for the solid-phase synthesis of 5'-triphosphate oligonucleotides is described. The full-length oligonucleotides are first constructed using standard solid-phase DNA/RNA synthesis, and then efficient implementation of a sequential 4-steps synthetic procedure, executed either manually or in a fully automated fashion, affords the corresponding solid-supported 5'-triphosphate oligonucleotides. Using this synthetic procedure, the full-length 5'-hydroxyl oligonucleotides are initially transformed into the corresponding 5'-H-phosphonate mono esters, subsequently oxidized in the presence of imidazole to the activated 5'-phosphorimidazolidates, and finally reacted with pyrophosphate on the solid support. The method uses safe, stable, and inexpensive reagents, and the process is scalable and readily applicable to automated synthesis compatible with the current commercially available DNA/RNA synthesizers. After cleavage from the solid support and deprotection, a range of DNA, RNA, and chemically modified 5'-triphosphate oligonucleotides are obtained in a convenient and efficient manner and isolated in good yields after HPLC purification.

PMID:
22956453
DOI:
10.1002/0471142700.nc0128s50
[Indexed for MEDLINE]

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