Fab Chaperone-Assisted Rna Crystallography (Fab Carc)

Abstract

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.

Publication Date

1-1-2016

Publication Title

Methods in molecular biology (Clifton, N.J.)

Volume

1320

Number of Pages

77-109

Document Type

Article

Personal Identifier

scopus

DOI Link

https://doi.org/10.1007/978-1-4939-2763-0_7

Socpus ID

84958621761 (Scopus)

Source API URL

https://api.elsevier.com/content/abstract/scopus_id/84958621761

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