Nucleic Acids Research Advance Access published online on January 26, 2007
Nucleic Acids Research, doi:10.1093/nar/gkl1110
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Methods online |
Reversible site-specific tagging of enzymatically synthesized RNAs using aldehyde–hydrazine chemistry and protease-cleavable linkers
ko I KirinInstitute of Pharmacy and Molecular Biotechnology, University of Heidelberg, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany
*To whom correspondence should be addressed. Tel: +49 6221 544851; Fax: +49 6221 546430. Email: jaeschke{at}uni-hd.de
Received November 6, 2006. Revised November 28, 2006. Accepted November 28, 2006.
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.