Synthetic antibodies for specific recognition and crystallization of structured RNA

Abstract

Antibodies that bind protein antigens are indispensable in biochemical research and modern medicine. However, knowledge of RNA-binding antibodies and their application in the ever-growing RNA field is lacking. Here we have developed a robust approach using a synthetic phage-display library to select specific antigenbinding fragments (Fabs) targeting a large functional RNA. We have solved the crystal structure of the first Fab-RNA complex at 1.95 Å. Capability in phasing and crystal contact formation suggests that the Fab provides a potentially valuable crystal chaperone for RNA. The crystal structure reveals that the Fab achieves specific RNA binding on a shallow surface with complementaritydetermining region (CDR) sequence diversity, length variability, and main-chain conformational plasticity. The Fab-RNA interface also differs significantly from Fab-protein interfaces in amino acid composition and light-chain participation. These findings yield valuable insights for engineering of Fabs as RNA-binding modules and facilitate further development of Fabs as possible therapeutic drugs and biochemical tools to explore RNA biology.antigen-binding fragments ͉ x-ray crystallography A ntibodies are integral components of the immune system and represent a rapidly growing sector of the biotechnology industry (1, 2). Clinically, antibodies serve as diagnostic markers for disease antigens and play increasingly important roles as therapeutic agents for a wide range of diseases (3). Antibodies also provide invaluable biomedical research tools, serving to define the components and functions of macromolecular complexes, to establish cellular distributions of proteins, and to facilitate structural analysis as chaperones for crystallization of membrane proteins (4-6). Hybridoma and other technologies have yielded antibodies against a vast array of specific antigens (2). An enormous body of literature documents the molecular details of antibody interactions with a variety of antigens, including proteins (7), polysaccharides (8), and small haptens (9). However, much less information (and, in particular, no structural information) exists for antibody-RNA interactions.The relative absence of antibodies that bind RNA from the immunologic repository is striking, especially considering that recent genome-wide analyses of the metazoan transcriptome have revealed the presence of vast numbers of noncoding RNAs, including silencing RNAs, riboswitches, catalytic RNAs, and a multitude of other functional RNA moleucles (10,11). A large number of these RNAs adopt complex three-dimensional architectures that frequently act in complex with proteins to mediate their biological function (12, 13). Nevertheless, with the exception of a handful of examples, mostly isolated from the sera of autoimmune patients (14-17), we know little about anti-RNA antibodies and their recognition of nucleic acids. This dearth of information reflects our inability to elicit antibodies against RNA by using traditional approaches. RNA appears to lack immunogenic potency...

NMR and XAS reveal an inner-sphere metal binding site in the P4 helix of the metallo-ribozyme ribonuclease P

“…The proposed model is strikingly similar to a metal site recently reported in the crystal structure of a group I intron fragment, in which the O6 and N7 moieties of guanosine form inner, and outer-sphere interactions, resp., with a hydrated magnesium ion (31). Furthermore, the recent structure of a truncated B. stearothermophilus RNase P RNA with the outer-sphere Mg 2þ mimic Os(III)hexamine revealed a metal binding site within 6 Å of the corresponding A3, A4, G6, G22, and G23 positions in the P4 helix mimic (18).…”

Improving small-angle X-ray scattering data for structural analyses of the RNA world

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Unwinding RNA's secrets: advances in the biology, physics, and modeling of complex RNAs

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