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.

The Determination of Quadruplex Structures

“…All approaches to quadruplex DNA and RNA crystallization, whether manual or automated, need to systematically screen a wide set of crystallizing conditions (Ducruix & Giegé, 1999;Campbell & Parkinson, 2007;Holbrook & Holbrook, 2010). It is now common practice to use automated robotic crystallization methods to rapidly set up and scan large numbers of crystallization trials, either in purely aqueous environments or under a thin layer of an oil.…”

HELIX: a new modular nucleic acid crystallization screen

“…Existing well defined methodologies include full-and partial-factorial experimental setups (Carter, 1979); sparse-matrix sampling, designed using randomly selected reagents from complete combinatorial matrices (Jancarik & Kim, 1991); orthogonal arrays, based on systematic combinations of reagents (Kingston et al, 1994); and reverse screening, employing a knowledge-based step-bystep protocol starting with a best guess (Stura et al, 1994). Out of these techniques, sparse-matrix sampling using commercially available crystallization screen kits has become a very popular way of determining the preliminary crystallization conditions for macromolecules (Holbrook & Holbrook, 2001), circumventing full sampling of the multidimensional condition space which can be expensive in time and resources. These pre-formulated kits are generally designed to match crystallization tray arrays, consisting of a screen of 24, 48 or 96 conditions, typically formulated around five combinations of buffers, more than ten different precipitants at two or three concentrations, and additionally, for nucleic acids, around five monovalent and five divalent cations.…”