title: A general and efficient approach for the construction of RNA oligonucleotides containing a 5'-phosphorothiolate linkage.
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-α-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.
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Chicago, 929 E. 57th Street, Room W406, Chicago, IL 60637, USA. nli@uchicago.edu
authors.forename: Nan-Sheng
authors.initials: NS
authors.lastname: Li
authors.forename: John K
authors.initials: JK
authors.lastname: Frederiksen
authors.forename: Selene C
authors.initials: SC
authors.lastname: Koo
authors.forename: Jun
authors.initials: J
authors.lastname: Lu
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Wilson
authors.forename: David M J
authors.initials: DM
authors.lastname: Lilley
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Piccirilli
mesh_terms: D009843:Oligoribonucleotides / Q000096:biosynthesis / Q000138:chemical synthesis / Q000737:chemistry*; D063065:Organophosphonates / Q000737:chemistry; D011130:RNA Ligase (ATP) / Q000378:metabolism; D016337:RNA, Catalytic / Q000378:metabolism; D013873:Thionucleotides / Q000096:biosynthesis / Q000138:chemical synthesis / Q000737:chemistry*
chemical_list: D009843:Oligoribonucleotides; D063065:Organophosphonates; D016337:RNA, Catalytic; D013873:Thionucleotides; D011130:RNA Ligase (ATP)