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 Aχ31 and U8-A14. Aχ31 was found to be bonded through NlH. The utility of C2 deuteration in nucleic acid NMR is thus demonstrated.