Procedure for C2 deuteration of nucleic acids and determination of Aψ31 psedouridine conformation by nuclear overhausere effect in yeast tRNA^Phe

Abstract

Nuclear Overhauser effect (NOE) combined with semispecific deuteration provides a general strategy for identification of exchangeable protons in nucleic base pairs, and has been extended to NOEs involving purine C2 protons in tRNA. Deuterated tri-ethyl orthoformate was condensed with 5(4)-aminoimidazole 4(5)-carboxamide to yield C2 deuterated hypoxanthine. C2 deuterated hypoxanthine was fed to a purine requiring mutant of yeast and C2 deuterated tRNA^Phe was isolated. This C2 deuterated tRNA^Phe was used to identify Aψ31 and U8-A14. Aψ31 was found to be bonded through NlH. The utility of C2 deuteration in nucleic acid NMR is thus demonstrated.