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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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Results of transcription experiments using hydrazine 1 as initiator. (A) Phosphorimage of preparative gel for the isolation of transcripts from transcription reactions performed in the presence of equal quantities of [α-P]-UTP and varying concentrations of hydrazine 1 with [GTP] = 1.25 mM. (B) Phosphorimage of streptavidin-dependent gel-shift assay for the determination of the level of incorporation of hydrazine 1 into transcripts from transcriptions with [GTP] = 1.25 mM. Prior to electrophoresis, transcripts were exposed to sulfo-NHS biotin. Approximately equal levels of radioactive material were loaded into each lane of the gel to facilitate quantitative analysis. (C) Combined yield and incorporation level data for transcriptions with hydrazine 1 using [GTP] = 1.25 mM where 100% represents the relative total RNA yield in the absence of 1 and the presence of 1.25 mM GTP. The proportion of hydrazine-initiated transcript is represented by the black area of each bar. (D) Combined yield and incorporation level data for transcriptions with hydrazine 1 using [GTP] = 0.32 mM where 100% represents the relative total RNA yield in the absence of 1 and the presence of 0.32 mM GTP. The proportion of hydrazine-initiated transcript within each experiment is represented by the black area.
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Figure 2: Results of transcription experiments using hydrazine 1 as initiator. (A) Phosphorimage of preparative gel for the isolation of transcripts from transcription reactions performed in the presence of equal quantities of [α-P]-UTP and varying concentrations of hydrazine 1 with [GTP] = 1.25 mM. (B) Phosphorimage of streptavidin-dependent gel-shift assay for the determination of the level of incorporation of hydrazine 1 into transcripts from transcriptions with [GTP] = 1.25 mM. Prior to electrophoresis, transcripts were exposed to sulfo-NHS biotin. Approximately equal levels of radioactive material were loaded into each lane of the gel to facilitate quantitative analysis. (C) Combined yield and incorporation level data for transcriptions with hydrazine 1 using [GTP] = 1.25 mM where 100% represents the relative total RNA yield in the absence of 1 and the presence of 1.25 mM GTP. The proportion of hydrazine-initiated transcript is represented by the black area of each bar. (D) Combined yield and incorporation level data for transcriptions with hydrazine 1 using [GTP] = 0.32 mM where 100% represents the relative total RNA yield in the absence of 1 and the presence of 0.32 mM GTP. The proportion of hydrazine-initiated transcript within each experiment is represented by the black area.

Mentions: Transcriptions to assess relative RNA yields in the presence of 5′-deoxy-5′-hydrazinylguanosine initiator 1 and its level of incorporation were performed in the presence of varying concentrations of 5′-deoxy-5′-hydrazinylguanosine 1 and [α-32P]-UTP using a known template dsDNA (ATRib, giving a 82 nt transcript).[29] The choice of this template was based on our previous successful use of this system with modified guanosine initiators.[26,27] Assessment of the total yield of RNA by phosphorimaging of a preparative urea-PAGE gel showed that increased concentrations of 5′-deoxy-5′-hydrazinylguanosine 1 did not deleteriously affect RNA yield (Figure 2).


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)

Results of transcription experiments using hydrazine 1 as initiator. (A) Phosphorimage of preparative gel for the isolation of transcripts from transcription reactions performed in the presence of equal quantities of [α-P]-UTP and varying concentrations of hydrazine 1 with [GTP] = 1.25 mM. (B) Phosphorimage of streptavidin-dependent gel-shift assay for the determination of the level of incorporation of hydrazine 1 into transcripts from transcriptions with [GTP] = 1.25 mM. Prior to electrophoresis, transcripts were exposed to sulfo-NHS biotin. Approximately equal levels of radioactive material were loaded into each lane of the gel to facilitate quantitative analysis. (C) Combined yield and incorporation level data for transcriptions with hydrazine 1 using [GTP] = 1.25 mM where 100% represents the relative total RNA yield in the absence of 1 and the presence of 1.25 mM GTP. The proportion of hydrazine-initiated transcript is represented by the black area of each bar. (D) Combined yield and incorporation level data for transcriptions with hydrazine 1 using [GTP] = 0.32 mM where 100% represents the relative total RNA yield in the absence of 1 and the presence of 0.32 mM GTP. The proportion of hydrazine-initiated transcript within each experiment is represented by the black area.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Results of transcription experiments using hydrazine 1 as initiator. (A) Phosphorimage of preparative gel for the isolation of transcripts from transcription reactions performed in the presence of equal quantities of [α-P]-UTP and varying concentrations of hydrazine 1 with [GTP] = 1.25 mM. (B) Phosphorimage of streptavidin-dependent gel-shift assay for the determination of the level of incorporation of hydrazine 1 into transcripts from transcriptions with [GTP] = 1.25 mM. Prior to electrophoresis, transcripts were exposed to sulfo-NHS biotin. Approximately equal levels of radioactive material were loaded into each lane of the gel to facilitate quantitative analysis. (C) Combined yield and incorporation level data for transcriptions with hydrazine 1 using [GTP] = 1.25 mM where 100% represents the relative total RNA yield in the absence of 1 and the presence of 1.25 mM GTP. The proportion of hydrazine-initiated transcript is represented by the black area of each bar. (D) Combined yield and incorporation level data for transcriptions with hydrazine 1 using [GTP] = 0.32 mM where 100% represents the relative total RNA yield in the absence of 1 and the presence of 0.32 mM GTP. The proportion of hydrazine-initiated transcript within each experiment is represented by the black area.
Mentions: Transcriptions to assess relative RNA yields in the presence of 5′-deoxy-5′-hydrazinylguanosine initiator 1 and its level of incorporation were performed in the presence of varying concentrations of 5′-deoxy-5′-hydrazinylguanosine 1 and [α-32P]-UTP using a known template dsDNA (ATRib, giving a 82 nt transcript).[29] The choice of this template was based on our previous successful use of this system with modified guanosine initiators.[26,27] Assessment of the total yield of RNA by phosphorimaging of a preparative urea-PAGE gel showed that increased concentrations of 5′-deoxy-5′-hydrazinylguanosine 1 did not deleteriously affect RNA yield (Figure 2).

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
Related in: MedlinePlus