Determining Structures of RNA Aptamers and Riboswitches by X-Ray Crystallography
Structural biology plays a central role in gaining a full understanding of the myriad roles of RNA in biology. In recent years, innovative approaches in RNA purification and crystallographic methods have lead to the visualization of an increasing number of unique structures, providing new insights into its function at the atomic level. This article presents general protocols which have streamlined the process of obtaining a homogeneous sample of properly folded and active RNA in high concentrations that crystallizes well in the presence of a suitable heavy-atom for phasing. Of particular importance are approaches toward RNA crystallography that include exploring “construct space” as opposed to “condition space”. Moreover, development of a highly flexible method for experimentally phasing RNA crystals may open the door to a relatively simple means of solving these structures.
Andrea L. Edwards and Andrew D. Garst contributed equally to this work.
Key wordsRNA purification RNA synthesis riboswitch aptamer RNA crystallography heavy-atom derivative
The contents of this work comprise much of the collected wisdom of a number of colleagues and members of the Batey laboratory. In particular, we would like to thank Jeffrey Kieft of the C.U. Health Sciences Center who has been instrumental in developing many of the ideas presented in this article. This work was made possible by a Research Scholar Grant from the American Cancer Society and support from the National Institutes of Health to R.T.B.
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