Unique structural and stabilizing roles for the individual pseudouridine residues in the 1920 region of Escherichia coli 23S rRNA

The synthesis of a 5'-O-BzH-2'-O-ACE-protected pseudouridine phosphoramidit e is reported [BzH, benzhydryloxy-bis(trimethylsilyloxy)sily; ACE, bis(2-ac etoxyethoxy)methyl]. The availability of the phosphoramidite allows for rel iable and efficient syntheses of hairpin RNAs containing single or multiple pseudouridine modifications in the stem or loop regions. Five 19-nt hairpi n RNAs representing the 1920-loop region (G(1906)-C-1924) of Escherichia co li 23S rRNA were synthesized with pseudouridine residues located at positio ns 1911, 1915 and 1917, Thermodynamic parameters, circular dichroism spectr a and NMR data are presented for all five RNAs, Overall, three different st ructural contexts for the pseudouridine residues were examined and compared with the unmodified RNA, Our main findings are that pseudouridine modifica tions exhibit a range of effects on RNA stability and structure, depending on their locations. More specifically, pseudouridines in the single-strande d loop regions of the model RNAs are slightly destabilizing, whereas a pseu douridine at the stem-loop junction is stabilizing. Furthermore, the observ ed effects on stability are approximately additive when multiple pseudourid ine residues are present. The possible relationship of these results to RNA function is discussed.