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Conformational analyses using the single-strand-specific nuclease from mung bean and restriction endonucleases have been performed on a series of DNA fragments related to the sequence of the yeast initiator tRNAMet. Mung bean nuclease cleaves DNA fragments exclusively in some, but not all, single-stranded regions as predicted by RNA secondary rules. Comparison of cleavage patterns of yeast initiator tRNAMet, tDNAMet (a DNA oligomer having the sequence of tRNAMet) and the anti-tDNAMet (the complement of tDNAMet) suggests that the conformation of the three molecules is very similar. Furthermore, both tDNA and anti-tDNA are cleaved by HhaI and CfoI restriction endonucleases at two GCG/C sites which would be in double- stranded regions (the acceptor and dihydrouridine stem), if the two molecules adopt the tRNA cloverleaf structure. On the other hand, minor cleavage products show that the core region, i.e. the extra loop area, is slightly more exposed in tDNA and in anti-tDNA than in tRNA. Therefore, we submit that the global conformation of nucleic acids is primarily dictated by the interaction of purine and pyrimidine bases with atoms and functional groups common to both RNA and DNA. In this view the 2′-hydroxyl group, in tRNA at least, is an auxiliary structural feature whose role is limited to fostering local interactions, which increase the stability of a given conformation.