title: Electrophilic activity-based RNA probes reveal a self-alkylating RNA for RNA labeling.
abstract: Probes that form covalent bonds with RNA molecules on the basis of their chemical reactivity would advance our ability to study the transcriptome. We developed a set of electrophilic activity-based RNA probes designed to react with unusually nucleophilic RNAs. We used these probes to identify reactive genome-encoded RNAs, resulting in the discovery of a 42-nt catalytic RNA from an archaebacterium that reacts with a 2,3-disubstituted epoxide at N7 of a specific guanosine. Detailed characterization of the catalytic RNA revealed the structural requirements for reactivity. We developed this catalytic RNA into a general tool to selectively conjugate a small molecule to an RNA of interest. This strategy enabled up to 500-fold enrichment of target RNA from total mammalian RNA or from cell lysate. We demonstrated the utility of this approach by selectively capturing proteins in yeast cell lysate that bind the ASH1 mRNA.
authors.affiliation: 1] Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA. [2] Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA.
authors.forename: Richard I
authors.initials: RI
authors.lastname: McDonald
authors.affiliation: 1] Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA. [2] Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA.
authors.forename: John P
authors.initials: JP
authors.lastname: Guilinger
authors.affiliation: 1] Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA. [2] Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA.
authors.forename: Shankar
authors.initials: S
authors.lastname: Mukherji
authors.affiliation: 1] Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA. [2] Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA.
authors.forename: Edward A
authors.initials: EA
authors.lastname: Curtis
authors.affiliation: 1] Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA. [2] Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA.
authors.forename: Won I
authors.identifier: 000000031451999X
authors.initials: WI
authors.lastname: Lee
authors.affiliation: 1] Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA. [2] Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA.
authors.forename: David R
authors.initials: DR
authors.lastname: Liu
mesh_terms: D000478:Alkylation; D001105:Archaea / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D002457:Cell Extracts / Q000737:chemistry; D004852:Epoxy Compounds / Q000737:chemistry; D006151:Guanosine / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D015347:RNA Probes / Q000138:chemical synthesis / Q000737:chemistry*; D016337:RNA, Catalytic / Q000737:chemistry*; D012333:RNA, Messenger / Q000737:chemistry* / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism; D052156:SELEX Aptamer Technique; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods*
chemical_list: C098733:ASH1 protein, S cerevisiae; D002457:Cell Extracts; D004852:Epoxy Compounds; D015347:RNA Probes; D016337:RNA, Catalytic; D012333:RNA, Messenger; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D006151:Guanosine