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

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.

FIGURE 1

5′-deoxy-5′-hydrazinylguanosine.

SCHEME 1.

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

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.

FIGURE 3

Predicted structure of the HAC1^u-based 3′-stem-loop of the Ire1p endoribonuclease substrate prior to fluorophore labeling. Emboldened letters represent conserved nucleotides in the CXGXXG site recognized by Ire1p. The arrow highlights the Ire1p cleavage site.

FIGURE 4.

Endoribonuclease activity assays at 31° C using FITC-labeled 5′-hydrazinyl-RNA transcripts based on the HAC1^u-based 3′-stem-loop of the Ire1p substrate. (A) Extended time-course demonstrating complete cleavage of the RNA substrate with [Ire1p] = 1 µM. (B) Initial rates time-course with [Ire1p] = 0.25 µM.