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

Abstract

Here 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 tRNA^Gly. 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 tRNA^Gly 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.

Keywords: Pseudouridine; RNA NMR; RNA solid phase synthesis; stable isotope labelling.