Antibodies as RNA crystallization chaperones.

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Bibliographic Details
Author / Creator:Huang, Hao.
Description:171 p.
Format: E-Resource Dissertations
Local Note:School code: 0330.
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Other authors / contributors:University of Chicago.
Notes:Advisor: Joseph A. Piccirilli.
Thesis (Ph.D.)--The University of Chicago, Division of the Physical Sciences, Department of Chemistry, 2015.
Dissertation Abstracts International, Volume: 76-08(E), Section: B.
Summary:Over the past few years, we have selected high affinity synthetic antigen binding fragment (Fab) binders against a diverse panel of structured RNA antigens by using M13 phage displayed libraries. In this work, RNA-specific Fabs have succeeded in chaperone-assisted RNA crystallography (CARC). By grafting a previously identified portable antibody-binding RNA hairpin epitope, we have determined the high-resolution crystal structure of Spinach, an RNA aptamer that binds a GFP-like ligand and activates its green fluorescence, with potentially widespread applications for in vivo labeling and imaging. Structures of Spinach both with and without bound fluorophore have been solved at 2.2 and 2.4 A resolution, respectively. It has an elongated structure containing two helical domains separated by an internal bulge that folds into a G-quadruplex motif of unusual topology. The G-quadruplex motif and adjacent nucleotides comprise a partially pre-formed binding site for the fluorophore. The fluorophore binds in a planar conformation and makes extensive aromatic stacking and hydrogen bond interactions with the RNA. These findings provide a foundation for structure-based engineering of new fluorophore-binding RNA aptamers. In addition, another case of antibody Fab specifically binding to a 12-nucleotide single-stranded RNA (ssRNA) is also presented in this work. It demonstrates that Fab binding stabilizes the ssRNA into a hairpin secondary structure with a short 2-base-pair stem, and results in the RNA sitting on the antibody CDR-H3 loop. This model of RNA recognition by an antibody is novel, and collectively with previous examples they shed light on engineering of RNA-specific antibody Fab libraries in future.