Reversible site-specific tagging of enzymatically synthesized RNAs using aldehyde-hydrazine chemistry and protease-cleavable linkers.
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.
Pfander
Stephanie
S
Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany.
Fiammengo
Roberto
R
Kirin
Srećko I
SI
Metzler-Nolte
Nils
N
Jäschke
Andres
A
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
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