A general and efficient approach for the construction of RNA oligonucleotides containing a 5'-phosphorothiolate linkage

Nan-Sheng Li, John K. Frederiksen, Selene C. Koo, Jun Lu, Timothy J. Wilson, David M. J. Lilley, Joseph A. Piccirilli

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    Abstract

    Oligoribonucleotides containing a 5'-phosphorothiolate linkage have provided effective tools to study the mechanisms of RNA catalysis, allowing resolution of kinetic ambiguity associated with mechanistic dissection and providing a strategy to establish linkage between catalysis and specific functional groups. However, challenges associated with their synthesis have limited wider application of these modified nucleic acids. Here, we describe a general semisynthetic strategy to obtain these oligoribonucleotides reliably and relatively efficiently. The approach begins with the chemical synthesis of an RNA dinucleotide containing the 5'-phosphorothiolate linkage, with the adjacent 2'-hydroxyl group protected as the photolabile 2'-O-o-nitrobenzyl or 2'-O-alpha-methyl-o-nitrobenzyl derivative. Enzymatic ligation of the 2'-protected dinucleotide to transcribed or chemically synthesized 5' and 3' flanking RNAs yields the full-length oligoribonucleotide. The photolabile protecting group increases the chemical stability of these highly activated oligoribonucleotides during synthesis and long-term storage but is easily removed with UV irradiation under neutral conditions, allowing immediate use of the modified RNA in biochemical experiments.

    Original languageEnglish
    Article numbere31
    Pages (from-to)-
    Number of pages16
    JournalNucleic Acids Research
    Volume39
    Issue number5
    DOIs
    Publication statusPublished - Mar 2011

    Keywords

    • METAL-ION CATALYSIS
    • TETRAHYMENA RIBOZYME REACTION
    • TRANSFER RIBONUCLEIC-ACIDS
    • ANTISENSE DRUG DESIGN
    • PRE-MESSENGER-RNA
    • GROUP-I RIBOZYME
    • HAMMERHEAD RIBOZYME
    • PROTECTING GROUPS
    • PREMESSENGER RNA
    • DIVALENT METAL

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