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Related Experiment Videos

Phosphorothioate substitution can substantially alter RNA conformation.

J S Smith1, E P Nikonowicz

  • 1Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77005, USA.

Biochemistry
|May 10, 2000
PubMed
Summary

Phosphorothioate substitution can alter RNA structure, challenging its use in studying RNA-ligand interactions. This method may change the molecule's conformation, affecting binding and catalysis studies.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Structural Biology

Background:

  • Phosphorothioate substitution-interference experiments are standard for mapping RNA-ligand interactions.
  • These experiments assume substitution doesn't affect RNA conformation.

Purpose of the Study:

  • To investigate the structural impact of phosphorothioate substitution on an RNA hairpin.
  • To validate the assumption that substitution maintains biological activity.

Main Methods:

  • Nuclear Magnetic Resonance (NMR) spectroscopy was employed.
  • Stereospecific phosphorothioate substitution was performed at five sites on an RNA hairpin.

Main Results:

  • Most substitutions caused minor backbone changes and increased nucleotide stacking.

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  • One substitution significantly altered RNA conformation, causing a key adenine to loop out.
  • This conformational change disrupted the binding site for bacteriophage MS2 capsid protein.

    • Conclusions:

    • Phosphorothioate substitution can substantially alter RNA conformation, especially in regions with irregular secondary structure.
    • The interpretation of substitution-interference experiments requires caution due to potential conformational changes.
    • This highlights limitations in using phosphorothioate substitutions to study RNA structure and function.