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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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Synthetis of acetal-protected aldehyde–guanosine phosphate 3. Reagents and conditions: (a) 2 (2.0 equiv), DCI (10 equiv), CH3CN, room temperature, 10 min; (b) TBHP (16 equiv), room temperature, 10 min, volatiles removal; (c) TBAF (50 equiv), THF, room temperature, overnight, 16% yield over three steps; (d) 2% TFA in water, room temperature, 15 min, complete deprotection.
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Figure 2: Synthetis of acetal-protected aldehyde–guanosine phosphate 3. Reagents and conditions: (a) 2 (2.0 equiv), DCI (10 equiv), CH3CN, room temperature, 10 min; (b) TBHP (16 equiv), room temperature, 10 min, volatiles removal; (c) TBAF (50 equiv), THF, room temperature, overnight, 16% yield over three steps; (d) 2% TFA in water, room temperature, 15 min, complete deprotection.

Mentions: Initiator nucleotide 3 was designed and synthesized to introduce an aldehyde function into RNA transcripts according to Figure 2. Commercial phosphoramidite 1 (22) was therefore coupled with readily available 2′,3′-O-silyl-protected guanosine 2 (26,27) by activation with 4,5-dicyanoimidazole, followed by oxidation and deprotection, leaving the acetal protecting group intact. After purification by reversed phase chromatography, 3 was isolated in 16% total yield, and the purity and identity established by HPLC and MALDI mass spectrometry.Figure 2.


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)

Synthetis of acetal-protected aldehyde–guanosine phosphate 3. Reagents and conditions: (a) 2 (2.0 equiv), DCI (10 equiv), CH3CN, room temperature, 10 min; (b) TBHP (16 equiv), room temperature, 10 min, volatiles removal; (c) TBAF (50 equiv), THF, room temperature, overnight, 16% yield over three steps; (d) 2% TFA in water, room temperature, 15 min, complete deprotection.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

Figure 2: Synthetis of acetal-protected aldehyde–guanosine phosphate 3. Reagents and conditions: (a) 2 (2.0 equiv), DCI (10 equiv), CH3CN, room temperature, 10 min; (b) TBHP (16 equiv), room temperature, 10 min, volatiles removal; (c) TBAF (50 equiv), THF, room temperature, overnight, 16% yield over three steps; (d) 2% TFA in water, room temperature, 15 min, complete deprotection.
Mentions: Initiator nucleotide 3 was designed and synthesized to introduce an aldehyde function into RNA transcripts according to Figure 2. Commercial phosphoramidite 1 (22) was therefore coupled with readily available 2′,3′-O-silyl-protected guanosine 2 (26,27) by activation with 4,5-dicyanoimidazole, followed by oxidation and deprotection, leaving the acetal protecting group intact. After purification by reversed phase chromatography, 3 was isolated in 16% total yield, and the purity and identity established by HPLC and MALDI mass spectrometry.Figure 2.

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