title: Reversible site-specific tagging of enzymatically synthesized RNAs using aldehyde-hydrazine chemistry and protease-cleavable linkers.
abstract: The investigation of RNA structure, dynamics and biological function often requires the site-specific incorporation of non-natural moieties. Here we describe the functionalization of RNA transcripts by aldehyde-hydrazine chemistry using a simple initiator nucleotide that carries an acetal-protected aldehyde function. This initiator nucleotide was efficiently incorporated into RNA, and the modified RNAs were quantitatively coupled to a peptide derivative displaying a hydrazine moiety at one end, a biotin tag at the other, and a trypsin-cleavable sequence in between. RNA conjugates could be easily isolated by affinity chromatography on streptavidin agarose and quantitatively cleaved off the support by trypsin treatment without detectable RNA degradation. The strategy described here may allow the incorporation of various new features into enzymatically synthesized RNA under mild conditions.
authors.affiliation: Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Pfander
authors.forename: Roberto
authors.initials: R
authors.lastname: Fiammengo
authors.forename: Srećko I
authors.initials: SI
authors.lastname: Kirin
authors.forename: Nils
authors.initials: N
authors.lastname: Metzler-Nolte
authors.forename: Andres
authors.initials: A
authors.lastname: Jäschke
mesh_terms: D000447:Aldehydes / Q000737:chemistry*; D001671:Biochemistry / Q000379:methods; D012321:DNA-Directed RNA Polymerases; D006834:Hydrazines / Q000737:chemistry*; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D012313:RNA / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D014357:Trypsin; D014764:Viral Proteins
chemical_list: D000447:Aldehydes; D006834:Hydrazines; D009711:Nucleotides; D010455:Peptides; D014764:Viral Proteins; D012313:RNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D014357:Trypsin