Thermodynamic contribution of nucleoside modifications to yeast tRNAPhe anticodon stem loop analogs

The determination of the structural and functional contributions of natural modified nucleosides to tRNA has been limited by lack of an approach that can systematically incorporate the modified units. We have produced a number of oligonucleotide analogs, or the anticodon of yeast tRNA(Phe) by, combining standard automated synthesis for the major nucleosides with specialty chemistries for the modified nucleosides. In this study, both naturally occurring and unnatural modified nucleotides were placed in native contexts. Each oligonucleotide was purified and the nucleoside composition determined to validate the chemistry. The RNAs were denatured and analyzed to determine the van't Hoff thermodynamic parameters. Here, we report the individual thermodynamic contributions for Cm, Gm, m(1)G, m(5)C, Psi. In addition m(5)m(6)U, m(1)Psi and m(3)Psi, introduced to gain additional understanding of the physicochemical contribution of Psi and m(5)C at an atomic level. These oligonucleotides demonstrate that modifications have measurable thermodynamic contributions and that loop modifications have global contributions.