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Reversible site-specific tagging of enzymatically synthesized RNAs using aldehyde-hydrazine chemistry and protease-cleavable linkers.

Pfander S, Fiammengo R, Kirin SI, Metzler-Nolte N, Jäschke A - Nucleic Acids Res. (2007)

Bottom Line: The investigation of RNA structure, dynamics and biological function often requires the site-specific incorporation of non-natural moieties.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany.

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.

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Site-specific tagging of RNA transcripts by hydrazone formation, immobilization of labelled RNA and subsequent proteolytic cleavage.
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Figure 1: Site-specific tagging of RNA transcripts by hydrazone formation, immobilization of labelled RNA and subsequent proteolytic cleavage.

Mentions: Aldehyde–amine and aldehyde–hydrazine coupling have successfully been used to modify oligonucleotides with many different functionalities, such as ligands for metal complexes, solid supports, amino-sugars or peptides (21,22). So far aldehyde groups have been selectively incorporated into long RNA transcripts only by post-transcriptional modification of a thiophosphate-modified RNA (23) or by periodate oxidation of the 3′-terminal ribose, which may result in unstable conjugates due to elimination reactions (24,25). In order to develop a simple aldehyde-based conjugation protocol we describe here the design and synthesis of an initiator nucleotide that carries a protected aldehyde function. We report the site-specific incorporation of this compound into RNA transcripts, including a 109 residue long RNA pool with 70 randomized positions, the efficient coupling of a biotinylated peptide using hydrazine chemistry, and the targeted release of the appended moieties by enzymatic cleavage (Figure 1).Figure 1.


Reversible site-specific tagging of enzymatically synthesized RNAs using aldehyde-hydrazine chemistry and protease-cleavable linkers.

Pfander S, Fiammengo R, Kirin SI, Metzler-Nolte N, Jäschke A - Nucleic Acids Res. (2007)

Site-specific tagging of RNA transcripts by hydrazone formation, immobilization of labelled RNA and subsequent proteolytic cleavage.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1851632&req=5

Figure 1: Site-specific tagging of RNA transcripts by hydrazone formation, immobilization of labelled RNA and subsequent proteolytic cleavage.
Mentions: Aldehyde–amine and aldehyde–hydrazine coupling have successfully been used to modify oligonucleotides with many different functionalities, such as ligands for metal complexes, solid supports, amino-sugars or peptides (21,22). So far aldehyde groups have been selectively incorporated into long RNA transcripts only by post-transcriptional modification of a thiophosphate-modified RNA (23) or by periodate oxidation of the 3′-terminal ribose, which may result in unstable conjugates due to elimination reactions (24,25). In order to develop a simple aldehyde-based conjugation protocol we describe here the design and synthesis of an initiator nucleotide that carries a protected aldehyde function. We report the site-specific incorporation of this compound into RNA transcripts, including a 109 residue long RNA pool with 70 randomized positions, the efficient coupling of a biotinylated peptide using hydrazine chemistry, and the targeted release of the appended moieties by enzymatic cleavage (Figure 1).Figure 1.

Bottom Line: The investigation of RNA structure, dynamics and biological function often requires the site-specific incorporation of non-natural moieties.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany.

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.

Show MeSH
Related in: MedlinePlus