Crystallisation of Nucleic Acids

AbstractHigh‐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 ofdeoxyribonucleic acid(DNA) orribonucleic 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.