Crystallisation of Nucleic Acids

Abstract

High‐resolution three dimensional structure analysis by X‐ray diffraction requires large, well‐ordered, single crystals. The crystallisation of nucleic acids has become the limiting step in their structural analysis by X‐ray crystallography. Nucleic acids may be isolated from native sources or synthesised chemically or enzymatically. Purification to homogeneity may be achieved by thin layer chromatography, polyacrylamide gel electrophoresis, column chromatography or combinations of these methods. Approaches to crystallisation have included batch methods, dialysis, evaporation, interface diffusion and vapour diffusion. The most widely and successfully used technique is hanging‐drop vapour diffusion. Sparse matrix screening has allowed exploration of the vast crystallisation space with the limited amount of nucleic acid available. Developments in synchrotron radiation and cryocrystallography have allowed use of smaller crystals for structure determination. High‐throughput robotic crystallisation uses less material thus allowing more crystallisation experiments from a given amount of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

Key Concepts:

  • The most complete structural description of a DNA or an RNA molecule is achieved through high resolution X‐ray crystallography.

  • Nucleic acid crystallisation depends on synthesis of large quantities of homogeneous DNA or RNA.

  • Material for crystallisation can be prepared by purification of native material, chemical synthesis or enzymatic synthesis.

  • Purification to homogeneity implies a single form of oligomeric state and modification in addition to a single species.

  • Purification methods include thin layer chromatography (TLC), polyacrylamide gel electrophoresis (PAGE) and liquid chromatography (LC).

  • The most widely used method for crystallisation is vapour diffusion in either a hanging drop or sitting drop format.

  • High‐throughput robotic crystallisation allows setup of a greater number of experiments while using less nucleic acid material.

  • The size of crystal needed for X‐ray diffraction analysis has been sharply reduced with the use of powerful synchrotron radiation and computer programs.

  • To date, over 2000 DNA and 1000 RNA crystal structures have been reported in public databases such as the Nucleic Acid Database and the Protein Data Bank. Most of these structures are nucleic acid complexed with protein or ligands.

Keywords: crystallisation; DNA; RNA; X‐ray crystallography; structure

Figure 1.

Schematic diagrams of (a) sitting drop and (b) hanging drop vapour diffusion crystallisation setups. The schematic shows a representation of one well in a crystallisation plate; plates typically come in 24 and 96 well formats. The drop contains the nucleic acid or protein of interest mixed with an equal volume of reservoir solution containing the precipitant (ppt). Water diffuses out of the drop, where the precipitant concentration is half that of the reservoir, effectively slowly concentrating the macromolecule of interest and promoting crystal growth. Reproduced from the Hampton Research Crystallization Tools Catalog. With permission from Hampton Research Corp.

Figure 2.

Some examples of RNA crystals. (a) A 69‐mer catalytic RNA, (b) a double helical portion of the rev responsive element, (c) tRNA and (d) an oligomer containing a mismatch. Note the plate‐ or flower‐like appearance, common to many RNA crystals. Reproduced from Holbrook SR (2001) Crystallization of RNA. Cellular and Molecular Life Sciences58(2): 234–243, with permission from Springer.

close

Further Reading

Baeyens KJ, Jancarik J and Holbrook SR (1994) Use of low‐molecular‐weight polyethylene glycol in the crystallization of RNA oligomers. Acta Crystallographica D50: 764–767.

Doudna JA, Grosshans C, Gooding A and Kundrot CE (1993) Crystallization of ribozymes and small RNA motifs by a sparse matrix approach. Proceedings of the National Academy of Sciences of the USA 90: 7829–7833.

Ducruix A and Geige R (eds) (1992) Crystallization of Nucleic Acids and Proteins – A Practical Approach. The Practical Approach Series. New York: IRL Press at Oxford University Press.

Golden BL and Kundrot CE (2003) RNA crystallization. Journal of Structural Biology 142: 98–107.

Holbrook SR, Holbrook EL and Walukiewicz HE (2000) Crystallization of RNA. CMLS, Cellular and Molecular Life Sciences 58: 234–243.

Holbrook SR and Kim S‐H (1997) RNA crystallography. Biopolymers 44: 3–21.

Ke A and Doudna J (2004) Crystallization of RNA and RNA‐protein complexes. Methods 34: 408–414.

Mooers BH (2009) Crystallogrphic studies of DNA and RNA. Methods 47: 168–176.

Scott WG, Finch JT, Grenfell R et al. (1995) Rapid crystallization of chemically synthesized hammerhead RNAs using a double screening procedure. Journal of Molecular Biology 250: 327–332.

Sproat BS (1993) Chemical nucleic acid synthesis, modification and labelling. Current Opinion in Biotechnology 4: 20–28.

Wahl MC, Ramakrishnan B, Ban C, Chen X and Sundaralingan M (1996) RNA – synthesis, purification and crystallization. Acta Crystallographica D52: 668–675.

Contact Editor close
Submit a note to the editor about this article by filling in the form below.

* Required Field

How to Cite close
Holbrook, Elizabeth L, and Holbrook, Stephen R(Sep 2010) Crystallisation of Nucleic Acids. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0002719.pub2]