NMR spectroscopy of RNA duplexes containing pseudouridine in supercooled water.
We have performed NMR experiments in supercooled water in order to decrease the temperature-dependent exchange of protons in RNA duplexes. NMR spectra of aqueous samples of RNA in bundles of narrow capillaries that were acquired at temperatures as low as -18 degrees C reveal resonances of exchangeable protons not seen at higher temperatures. In particular, we detected the imino protons of terminal base pairs and the imino proton of a non-base-paired pseudouridine in a duplex representing the eukaryotic pre-mRNA branch site helix. Analysis of the temperature dependence of chemical shift changes (thermal coefficients) for imino protons corroborated hydrogen bonding patterns observed in the NMR-derived structural model of the branch site helix. The ability to observe non-base-paired imino protons of RNA is of significant value in structure determination of RNA motifs containing loop and bulge regions.
Department of Chemistry and Biochemistry, Dittmer Laboratory of Chemistry, Florida State University, Tallahassee, FL 32306-4390, USA.
Kersten T
KT
Schroeder
Jack J
JJ
Skalicky
Nancy L
NL
Greenbaum
D001483:Base Sequence; D003080:Cold Temperature; D006860:Hydrogen Bonding; D008956:Models, Chemical; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D009690:Nucleic Acid Conformation; D011522:Protons; D011560:Pseudouridine / Q000737:chemistry*; D012313:RNA / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D014867:Water / Q000737:chemistry*
D011522:Protons; D014867:Water; D011560:Pseudouridine; D012313:RNA