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Related Experiment Videos

RNA - synthesis, purification and crystallization.

M C Wahl1, B Ramakrishnan, C Ban

  • 1The Ohio State University, Laboratory of Biological Macromolecular Structure, Department of Biochemistry, Columbus 43210-1002, USA.

Acta Crystallographica. Section D, Biological Crystallography
|July 1, 1996
PubMed
Summary

This study presents reliable protocols for synthesizing and purifying RNA and DNA-RNA chimeras for X-ray crystallography. These methods enable efficient screening and optimization for successful crystallization, yielding high-resolution diffraction data.

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Area of Science:

  • Biochemistry and Molecular Biology
  • Structural Biology
  • Chemical Synthesis

Background:

  • X-ray crystallography requires highly pure nucleic acid samples.
  • Obtaining suitable crystals for RNA and DNA-RNA chimeras has been challenging.
  • Standardized protocols are needed for reproducible structural studies.

Purpose of the Study:

  • To describe routine protocols for chemical synthesis and purification of RNA and DNA-RNA chimeras.
  • To present an efficient screening and optimization protocol for crystallizing these molecules.
  • To enable researchers to reproduce these methods for structural determination.

Main Methods:

  • Chemical synthesis of milligram quantities of RNA and DNA-RNA chimeras.
  • Purification techniques tailored for crystallographic demands.

Related Experiment Videos

  • Screening protocols to assess crystallizability.
  • Optimization of crystallization conditions, adapting DNA oligomer methods for RNA.

    • Main Results:

    • Successful synthesis and purification of various RNA and DNA-RNA chimera sequences.
    • Established crystallization conditions applicable to alternating octamers, G/C-rich decamers, and sequences with overhangs.
    • Demonstrated successful crystallization of drug complexes with chimeras.
    • Obtained crystals diffracting to high resolution, validating the protocols.

    Conclusions:

    • The described protocols facilitate routine production and crystallization of RNA and DNA-RNA chimeras.
    • Crystallization conditions for RNA are similar to those for DNA oligomers.
    • These methods are robust, enabling high-resolution structural studies of diverse nucleic acid constructs and their complexes.