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5'-deoxy-5'-hydrazinylguanosine as an initiator of T7 Rna polymerase-catalyzed transcriptions for the preparation of labeling-ready RNAs.

Skipsey M, Hack G, Hooper TA, Shankey MC, Conway LP, Schröder M, Hodgson DR - Nucleosides Nucleotides Nucleic Acids (2013)

Bottom Line: 5'-deoxy-5'-hydrazinylguanosine was incorporated into the 5'-termini of RNA transcripts using T7 RNA polymerase.Transcriptions provided 5'-hydrazinyl-RNA that was readily labeled and purified.The use of fluorophore-labeled material was validated in an endoribonuclease activity assay.

View Article: PubMed Central - PubMed

Affiliation: a Department of Chemistry and Biophysical Sciences Institute , Durham University , Durham , United Kingdom.

ABSTRACT
5'-deoxy-5'-hydrazinylguanosine was incorporated into the 5'-termini of RNA transcripts using T7 RNA polymerase. Transcriptions provided 5'-hydrazinyl-RNA that was readily labeled and purified. The use of fluorophore-labeled material was validated in an endoribonuclease activity assay.

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Model reaction to estimate the modification of the 5′-hydrazinyl functional group with FITC.
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SCH1: Model reaction to estimate the modification of the 5′-hydrazinyl functional group with FITC.

Mentions: 5′-deoxy-5′-hydrazinylguanosine 1 (15 mg, 50 µmol) was dissolved in triethylammonium bicarbonate (TEAB) solution (1 M, 22.5 ml, pH 8) and cooled in an ice bath. A solution of FITC (97 mg, 270 µmol) in dry DMF (2.5 ml) was added to the reaction vessel dropwise over the course of 10 minutes. The reaction mixture was left to stir on ice for 4 hours before being lyophilized. The residue was then dissolved in ammonia solution (2.5 ml, 35% w/v), the mixture was stirred for 4 hours at room temperature, diluted with water (10 ml) and lyophilized. The resulting solid residue was then purified by ion exchange chromatography on a DEAE Sepharose Fast Flow™anion exchange resin (26 mm i.d. × 80 mm height) connected to an AKTAprime™plus chromatography system. The TEAB buffer gradient was constructed to rise from 0 M to 1 M over 200 minutes, with a flow rate of 4 ml/minute and detection at 254 nm. A control experiment was performed under identical conditions, in the absence of 5′-deoxy-5′-hydrazinylguanosine 1. Residues from the control were also chromatographed, and the chromatograms for reactions with and without 5′-deoxy-5′-hydrazinylguanosine 1 were compared. In the presence of 5′-deoxy-5′-hydrazinylguanosine 1, another species was apparent in the chromatogram, and this material was isolated by lyophilization, and analyzed by MS. The MS analysis confirmed the identity of this species as the adduct between FITC and 5′-deoxy-5′-hydrazinylguanosine 1, namely thiosemicarbazide 2 (see Scheme 1 for details). We observed no evidence for the formation of bis-FITC–labeled products.


5'-deoxy-5'-hydrazinylguanosine as an initiator of T7 Rna polymerase-catalyzed transcriptions for the preparation of labeling-ready RNAs.

Skipsey M, Hack G, Hooper TA, Shankey MC, Conway LP, Schröder M, Hodgson DR - Nucleosides Nucleotides Nucleic Acids (2013)

Model reaction to estimate the modification of the 5′-hydrazinyl functional group with FITC.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3924349&req=5

SCH1: Model reaction to estimate the modification of the 5′-hydrazinyl functional group with FITC.
Mentions: 5′-deoxy-5′-hydrazinylguanosine 1 (15 mg, 50 µmol) was dissolved in triethylammonium bicarbonate (TEAB) solution (1 M, 22.5 ml, pH 8) and cooled in an ice bath. A solution of FITC (97 mg, 270 µmol) in dry DMF (2.5 ml) was added to the reaction vessel dropwise over the course of 10 minutes. The reaction mixture was left to stir on ice for 4 hours before being lyophilized. The residue was then dissolved in ammonia solution (2.5 ml, 35% w/v), the mixture was stirred for 4 hours at room temperature, diluted with water (10 ml) and lyophilized. The resulting solid residue was then purified by ion exchange chromatography on a DEAE Sepharose Fast Flow™anion exchange resin (26 mm i.d. × 80 mm height) connected to an AKTAprime™plus chromatography system. The TEAB buffer gradient was constructed to rise from 0 M to 1 M over 200 minutes, with a flow rate of 4 ml/minute and detection at 254 nm. A control experiment was performed under identical conditions, in the absence of 5′-deoxy-5′-hydrazinylguanosine 1. Residues from the control were also chromatographed, and the chromatograms for reactions with and without 5′-deoxy-5′-hydrazinylguanosine 1 were compared. In the presence of 5′-deoxy-5′-hydrazinylguanosine 1, another species was apparent in the chromatogram, and this material was isolated by lyophilization, and analyzed by MS. The MS analysis confirmed the identity of this species as the adduct between FITC and 5′-deoxy-5′-hydrazinylguanosine 1, namely thiosemicarbazide 2 (see Scheme 1 for details). We observed no evidence for the formation of bis-FITC–labeled products.

Bottom Line: 5'-deoxy-5'-hydrazinylguanosine was incorporated into the 5'-termini of RNA transcripts using T7 RNA polymerase.Transcriptions provided 5'-hydrazinyl-RNA that was readily labeled and purified.The use of fluorophore-labeled material was validated in an endoribonuclease activity assay.

View Article: PubMed Central - PubMed

Affiliation: a Department of Chemistry and Biophysical Sciences Institute , Durham University , Durham , United Kingdom.

ABSTRACT
5'-deoxy-5'-hydrazinylguanosine was incorporated into the 5'-termini of RNA transcripts using T7 RNA polymerase. Transcriptions provided 5'-hydrazinyl-RNA that was readily labeled and purified. The use of fluorophore-labeled material was validated in an endoribonuclease activity assay.

Show MeSH