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

    Research output: Contribution to journalArticle

    10 Citations (Scopus)

    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

    Cite this

    Li, Nan-Sheng ; Frederiksen, John K. ; Koo, Selene C. ; Lu, Jun ; Wilson, Timothy J. ; Lilley, David M. J. ; Piccirilli, Joseph A. / A general and efficient approach for the construction of RNA oligonucleotides containing a 5'-phosphorothiolate linkage. In: Nucleic Acids Research. 2011 ; Vol. 39, No. 5. pp. -.
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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.",
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    A general and efficient approach for the construction of RNA oligonucleotides containing a 5'-phosphorothiolate linkage. / Li, Nan-Sheng; Frederiksen, John K.; Koo, Selene C.; Lu, Jun; Wilson, Timothy J.; Lilley, David M. J.; Piccirilli, Joseph A.

    In: Nucleic Acids Research, Vol. 39, No. 5, e31, 03.2011, p. -.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - A general and efficient approach for the construction of RNA oligonucleotides containing a 5'-phosphorothiolate linkage

    AU - Li, Nan-Sheng

    AU - Frederiksen, John K.

    AU - Koo, Selene C.

    AU - Lu, Jun

    AU - Wilson, Timothy J.

    AU - Lilley, David M. J.

    AU - Piccirilli, Joseph A.

    PY - 2011/3

    Y1 - 2011/3

    N2 - 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.

    AB - 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.

    KW - METAL-ION CATALYSIS

    KW - TETRAHYMENA RIBOZYME REACTION

    KW - TRANSFER RIBONUCLEIC-ACIDS

    KW - ANTISENSE DRUG DESIGN

    KW - PRE-MESSENGER-RNA

    KW - GROUP-I RIBOZYME

    KW - HAMMERHEAD RIBOZYME

    KW - PROTECTING GROUPS

    KW - PREMESSENGER RNA

    KW - DIVALENT METAL

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    DO - 10.1093/nar/gkq1265

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    SP - -

    JO - Nucleic Acids Research

    JF - Nucleic Acids Research

    SN - 0305-1048

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    M1 - e31

    ER -