Nucleic Acids Research, 1995, Vol. 23, No. 24 5020-5026
© 1995
Articles |
Stabilization of RNA stacking by pseudouridine
Department of Medicinal Chemistry, University of Utah Salt Lake City, UT 84112, USA
Received September 12, 1995. Accepted November 16, 1995.
The effect of the modified nucleoslde pseudouridine (
) on RNA structure was compared with uridine. The extent of base stacking in model RNA oligonucleotides was measured by 1H NMR, UV, and CD spectroscopy. The UV and CD results Indicate that the model single-stranded oligoribonucleotides AAUA and AATA form stacked structures in solution and the CD results for AAA are consistent with a general A-form helical conformation. The AAA oligomer exhibits a greater degree of UV hypochromlcity over the temperature range 5-55 °C, consistent with a better stacked, more A-form structure compared with AAUA. The extent of stacking for each nucleotide residue was inferred from the percent 3'-endo sugar conformation as indicated by the H1'-H2' NMR scalar coupling. This indirect indication of stacking was confirmed by sequential NOE connectivity patterns obtained from 2D ROESY NMR experiments. NMR measurements as a function of temperature indicate that pseudouridine forms a more stable base stacking arrangement than uridine, an effect that is propagated throughout the helix to stabilize stacking of neighboring purine nucleosides. The N1-H Imino proton in AAA exchanges slowly with solvent, suggesting a role for the extra imino proton in stabilizing the conformation of pseudouridine. These results show that the conformational stabilization is an intrinsic property of pseudouridine occurring at the nucleotide level. The characteristics of pseudouridine in these models are consistent with earlier studies on intact tRNA, indicating that pseudouridine probably performs the same stabilizing function in most structural contexts.
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