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.
journal_name
Nucleic Acids Resjournal_title
Nucleic acids researchauthors
Li NS,Frederiksen JK,Koo SC,Lu J,Wilson TJ,Lilley DM,Piccirilli JAdoi
10.1093/nar/gkq1265subject
Has Abstractpub_date
2011-03-01 00:00:00pages
e31issue
5eissn
0305-1048issn
1362-4962pii
gkq1265journal_volume
39pub_type
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