Preparation and crystallization of RNA

Abstract

The field of RNA structure has exploded in recent years, in part owing to advances in crystallography of RNA molecules. This phenomenon can largely be attributed to the development of three modern methods: (1) large-scale in vitro RNA synthesis, (2) cryocrystallography, and (3) high-intensity synchrotron beamlines. Milligram quantities of RNA can be routinely synthesized using either chemical or enzymatic syntheses, making it feasible to carry out routine crystallization experiments on RNA. This has allowed crystals of RNA to be readily obtained. Generally, RNA crystals tend to be susceptible to radiation damage and to diffract X-rays more weakly than their protein counterparts. However, cryocrystallography and the high-intensity X-ray sources have overcome many of the difficulties involved in solving crystal structures of RNA. As a result of these advances, we now have a database of RNA structures that span from simple duplexes and hairpins to complex ribozymes and ribosomes. The protocols presented here describe methods to synthesize, purify, crystallize, and derivatize RNA for use in crystallographic studies.