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...

RNA crystallization and phasing represent major bottlenecks in RNA structure determination. Seeking to exploit antibody fragments as RNA crystallization chaperones, we have used an arginine-enriched synthetic Fab library displayed on phage to obtain Fabs against the class I ligase ribozyme. We solved the structure of a Fab:ligase complex at 3.1Å using molecular replacement with Fab coordinates, confirming the ribozyme architecture and revealing the chaperone’s role in RNA recognition and crystal contacts. The epitope resides in the GAAACAC sequence that caps the P5 helix and retains high-affinity Fab binding within the context of other structured RNAs. This portable epitope provides a new RNA crystallization chaperone system that easily can be screened in parallel to the U1A RNA-binding protein, with the advantages of the smaller size of the loop and high molecular weight, large surface area, and phasing power provided by Fabs.

Site-directed spin labeling with continuous wave electron paramagnetic resonance (EPR) spectroscopy was utilized to characterize dynamic features of the kink–turn motif formed through a leader–linker interaction in the Vibrio cholerae glycine riboswitch. Efficient incorporation of spin-labels into select sites within the phosphate backbone of the leader–linker region proceeded via splinted ligation of chemically synthesized spin-labeled oligonucleotides to in vitro transcribed larger RNA fragments. The resultant nitroxide EPR line shapes have spectral characteristics consistent with a kink–turn motif and reveal differential backbone dynamics that are modulated by the presence of magnesium, potassium, and glycine.

A closer look at RNA cleavage: A series of fluorine‐bearing nucleotide analogues (X) were incorporated into RNA to perturb the pKa value of the 2′‐OH group systematically. These analogues enable the use of physical organic approaches to investigate bonding to the nucleophile during the RNA scission reaction. Measurement of the Brønsted coefficient, βnuc, provides new insights into the transition‐state structure for this classic reaction.