5′-Deoxy-5′-Hydrazinylguanosine as an Initiator of T7 Rna Polymerase-Catalyzed Transcriptions for the Preparation of Labeling-Ready RNAs

Skipsey, Mark; Hack, Gordon; Hooper, Thomas A.; Shankey, Mark C.; Conway, Louis P.; Schröder, Martin; Hodgson, David R. W.

doi:10.1080/15257770.2013.851393

Cited by 7 publications

(2 citation statements)

References 37 publications

(45 reference statements)

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“…These findings with az G parallel those obtained when th G was the G analogue, and underscore the tolerance of T7RNAP P266L to accept two different G analogues. Our work should motivate studies to test other G analogues (especially non‐azide/alkyne initiators such as 5′‐hydrazinyl‐5′‐deoxyguanosine), and determine whether a common 10‐nt leader/initiating sequence rich in A, C, or U would afford high yields of RNAs initiated with different G analogues. The use of programmable RNases (e.g., Argonautes loaded with defined DNA guides) for site‐specific cleavage of large RNAs and the generation of smaller cleavage products bearing the 5′‐G analogue initiator also merits consideration.…”

Section: Figurementioning

confidence: 99%

An RNase P‐Based Assay for Accurate Determination of the 5′‐Deoxy‐5′‐azidoguanosine‐Modified Fraction of in Vitro‐Transcribed RNAs

et al. 2018

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Chemoenzymatic approaches are important for generating site-specific, chemically modified RNAs, a cornerstone for RNA structure-function correlation studies. T7 RNA polymerase (T7RNAP)-mediated in vitro transcription (IVT) of a DNA template containing the G-initiating class III Φ6.5 promoter is typically used to generate 5'-chemically modified RNAs by including a guanosine analogue (G analogue) initiator in the IVT. However, the yield of 5'-G analogue-initiated RNA is often poor and variable due to the high ratios of G analogue:GTP used in IVT. We recently reported that a T7RNAP P266L mutant afforded an approximately three-fold increase in fluorescent 5'-thienoguanosine-initiated pre-tRNA compared to the wild type T7RNAP. We have further explored the use of T7RNAP P266L to generate 5'-deoxy-5'-azidoguanosine ( G)-initiated RNA and found that the mutant yielded approximately four times more G-initiated pre-tRNA than the wild type in an IVT containing a 10:1 ratio of G:GTP. For accurate quantitation of the 5'- G-initiated RNA fraction, we employed RNase P, an endonuclease that catalyzes the removal of the 5'-leader in pre-tRNAs. Importantly, we show herein how RNase P can be leveraged for assessing 5'-G analogue incorporation in any RNA by rendering the target RNA, upon its binding to a customized external guide sequence RNA, into an unnatural substrate of RNase P. Such an approach in conjunction with T7RNAP P266L-based IVT should aid chemoenzymatic methods that are designed to generate 5'-chemically modified RNAs.

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Section: Figurementioning

confidence: 99%

An RNase P‐Based Assay for Accurate Determination of the 5′‐Deoxy‐5′‐azidoguanosine‐Modified Fraction of in Vitro‐Transcribed RNAs

et al. 2018

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“…5,6 Modied nucleosides and nucleotides also offer reactive functionalites for bioconjugation. [7][8][9][10] Standard methods for the synthesis of nucleoside phosphates, such as the Poulter 11 and Yoshikawa 12 syntheses, are frequently cumbersome, and require rigorously dry conditions and laborious purication. The use of the phosphoramidite method, while highly efficient for the synthesis of oligonucleotides when the protected nucleoside phosphoramidites are commercially available, becomes more laborious and requires global protection and rigorously dry conditions where the de novo syntheses of non-standard phosphoramidites are required.…”

Section: Introductionmentioning

confidence: 99%

The aqueous N-phosphorylation and N-thiophosphorylation of aminonucleosides

et al. 2014

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We demonstrate N-phosphorylation and N-thiophosphorylation of unprotected aminonucleosides in aqueous media. N-Phosphorylations using phosphoric chloride and N-thiophosphorylations using thiophosphoryl chloride were explored as functions of pH using 5 0-amino-5 0-deoxyguanosine as substrate. These reagents were compared to phosphodichloridate and thiophosphodichloridate ions, and the methodology was applied to other aminonucleosides. S-Alkylations of the nucleoside N-thiophosphoramidates were investigated as functions of pH and alkylating agent.

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A T7 RNA Polymerase Mutant Enhances the Yield of 5′‐Thienoguanosine‐Initiated RNAs

Lyon

Gopalan

2017

ChemBioChem

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Spectroscopic methods, which are used to establish RNA structure-function relationships, require strategies for post-synthetic, site-specific incorporation of chemical probes into target RNAs. For RNAs larger than 50 nt, the enzymatic incorporation of a nucleoside or nucleotide monophosphate guanosine analogue (G analogue) at their 5'-end is routinely achieved by T7 RNA polymerase (T7RNAP)-mediated in vitro transcription (IVT) of the appropriate DNA template containing a GTP-initiating class III Φ6.5 promoter. However, when high G analogue:GTP ratios are used to bias G analogue incorporation at the 5'-end, RNA yield is compromised. Here, we show that the use of a T7RNAP P266L mutant in IVT with 10:1 thienoguanosine ( G):GTP increased the percent incorporation and yield of 5'- G-initiated precursor tRNA for a net ≈threefold gain compared to IVT with wild-type T7RNAP. We also demonstrated that a one-pot multienzyme approach, consisting of transcription by T7RNAP P266L and post-transcriptional cleanup by polyphosphatase and an exonuclease, led to essentially near-homogeneous 5'- G-modified transcripts. This approach should be of broad utility in preparing 5'-modified RNAs.

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5′-Deoxy-5′-Hydrazinylguanosine as an Initiator of T7 Rna Polymerase-Catalyzed Transcriptions for the Preparation of Labeling-Ready RNAs

Abstract: 2 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.

Cited by 7 publications

References 37 publications

An RNase P‐Based Assay for Accurate Determination of the 5′‐Deoxy‐5′‐azidoguanosine‐Modified Fraction of in Vitro‐Transcribed RNAs

An RNase P‐Based Assay for Accurate Determination of the 5′‐Deoxy‐5′‐azidoguanosine‐Modified Fraction of in Vitro‐Transcribed RNAs

The aqueous N-phosphorylation and N-thiophosphorylation of aminonucleosides

A T7 RNA Polymerase Mutant Enhances the Yield of 5′‐Thienoguanosine‐Initiated RNAs

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