Efficient Synthetic Access to Stable Isotope Labelled Pseudouridine Phosphoramidites for RNA NMR Spectroscopy

Author:

Glänzer David1,Pfeiffer Martin23,Ribar Andrej23,Zeindl Ricarda1,Tollinger Martin1,Nidetzky Bernd23,Kreutz Christoph1ORCID

Affiliation:

1. Institute of Organic Chemistry and Center for Molecular Biosciences Innsbruck (CMBI) University of Innsbruck Innrain 80/82 6020 Innsbruck Austria

2. Institute of Biotechnology and Biochemical Engineering Graz University of Technology NAWI Graz Petersgasse 12 A-8010 Graz, Austria

3. and Austrian Centre of Industrial Biotechnology (acib) Krenngasse 37 A-8010 Graz Austria

Abstract

AbstractHere we report the efficient synthetic access to 13C/15N‐labelled pseudouridine phosphoramidites, which were incorporated into a binary H/ACA box guide RNA/product complex comprising 77 nucleotides (nts) in total and into a 75 nt E. coli tRNAGly. The stable isotope (SI) labelled pseudouridines were produced via a highly efficient chemo‐enzymatic synthesis. 13C/15N labelled uracils were produced via chemical synthesis and enzymatically converted to pseudouridine 5′‐monophosphate (ΨMP) by using YeiN, a Ψ‐5′‐monophosphate C‐glycosidase. Removal of the 5′‐phosphate group yielded the desired pseudouridine nucleoside (Ψ), which was transformed into a phosphoramidite building suitable for RNA solid phase synthesis. A Ψ ‐building block carrying both a 13C and a 15N label was incorporated into a product RNA and the complex formation with a 63 nt H/ACA box RNA could be observed via NMR. Furthermore, the SI labelled pseudouridine building block was used to determine imino proton bulk water exchange rates of a 75 nt E. coli tRNAGly CCmnm5U, identifying the TΨC‐loop 5‐methyluridine as a modifier of the exchange rates. The efficient synthetic access to SI‐labelled Ψ building blocks will allow the solution and solid‐state NMR spectroscopic studies of Ψ containing RNAs and will facilitate the mass spectrometric analysis of Ψ‐modified nucleic acids.

Funder

Austrian Science Fund

Österreichische Forschungsförderungsgesellschaft

Publisher

Wiley

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