Enhanced NMR signal detection of imino protons in RNA molecules containing 3′ dangling nucleotides
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We present a method for improving the quality of nuclear magnetic resonance (NMR) spectra involving exchangeable protons near the base of the stem of RNA hairpin molecules. NMR spectra of five different RNA hairpins were compared. These hairpins consisted of a native RNA structure and four molecules each having different unpaired, or dangling, nucleotides at the 3′ end. NMR experiments were acquired in water for each construct and the quality of the imino proton spectral regions were examined. The imino resonances near the base of the stem of the wild type RNA structure were not observed due to breathing motions. However, a significant increase in spectral quality for molecules with dangling 3′ adenosine or guanosine nucleotides was observed, with imino protons detected in these constructs that were not observed in the wild type construct. A modest improvement in spectral quality was seen for the construct with a 3′ unpaired uridine, whereas no significant improvement was observed for a 3′ unpaired cytidine. This improvement in NMR spectral quality mirrors the increased thermodynamic stability observed for 3′ unpaired nucleotides which is dependant on the stacking interactions of these nucleotides against the base of the stem. The use of a dangling 3′ adenosine nucleotide represents an easy method to significantly improve the quality of NMR spectra of RNA molecules.
KeywordsNMR spectroscopy RNA NMR RNA stability RNA structure
We thank Thomas Shrader (Department of Biochemistry, Albert Einstein College of Medicine) for the gift of the expression plasmid containing the histidine-tagged T7 RNA polymerase and Alex Beribisky and Tony Tavares for critical reading of this manuscript. This work was supported by funding from the Natural Sciences and Engineering Research Council of Canada (NSERC) to PEJ.
- Huttenhofer A, Westhof E, Böck A (1996) Solution structure of mRNA hairpins promoting selenocysteine incorporation in Escherichia coli and their base-specific interaction with special elongation factor SelB. RNA 2:354–366Google Scholar
- Neilson T, Romaniuk PJ, Alkema D, Hughes DW, Everett JR, Bell RA (1980) The effects of base sequence and dangling bases on the stability of short ribonucleic acid duplexes. Nucleic Acids Symp. Ser 293–311Google Scholar
- Puglisi EV, Puglisi JD (1998) Nuclear Magnetic Resonance of RNA. In: Simons RW, Grunberg-Manago M (eds) RNA structure and function, Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
- Wyatt JR, Chastain M, Puglisi JD (1991) Synthesis and purification of large amounts of RNA oligonucleotides. Biotechniques 11:764–769Google Scholar