Fab Chaperone-Assisted RNA Crystallography (Fab CARC)
Recent discovery of structured RNAs such as ribozymes and riboswitches shows that there is still much to learn about the structure and function of RNAs. Knowledge learned can be employed in both biochemical research and clinical applications. X-ray crystallography gives unparalleled atomic-level structural detail from which functional inferences can be deduced. However, the difficulty in obtaining high-quality crystals and their phasing information make it a very challenging task. RNA crystallography is particularly arduous due to several factors such as RNA’s paucity of surface chemical diversity, lability, repetitive anionic backbone, and flexibility, all of which are counterproductive to crystal packing. Here we describe Fab chaperone assisted RNA crystallography (CARC), a systematic technique to increase RNA crystallography success by facilitating crystal packing as well as expediting phase determination through molecular replacement of conserved Fab domains. Major steps described in this chapter include selection of a synthetic Fab library displayed on M13 phage against a structured RNA crystallization target, ELISA for initial choice of binding Fabs, Fab expression followed by protein A affinity then cation exchange chromatography purification, final choice of Fab by binding specificity and affinity as determined by a dot blot assay, and lastly gel filtration purification of a large quantity of chosen Fabs for crystallization.
Key wordsRNA crystallography Chaperone assisted crystallography Fab (antigen binding fragment) Phage display selection
- 6.Nakatani J, Tamada K, Hatanaka F, Ise S, Ohta H, Inoue K, Tomonaga S, Watanabe Y, Chung YJ, Banerjee R, Iwamoto K, Kato T, Okazawa M, Yamauchi K, Tanda K, Takao K, Miyakawa T, Bradley A, Takumi T (2009) Abnormal behavior in a chromosome-engineered mouse model for human 15q11-13 duplication seen in autism. Cell 137:1235–1246PubMedCentralPubMedCrossRefGoogle Scholar
- 31.Fellouse FA, Esaki K, Birtalan S, Raptis D, Cancasci VJ, Koide A, Jhurani P, Vasser M, Wiesmann C, Kossiakoff AA, Koide S, Sidhu SS (2007) High-throughput generation of synthetic antibodies from highly functional minimalist phage-displayed libraries. J Mol Biol 373:924–940PubMedCrossRefGoogle Scholar