Specific RNA-Binding Antibodies with a Four-Amino-Acid Code |
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Authors: | Eileen M. Sherman Sean HolmesJing-Dong Ye |
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Affiliation: | Department of Chemistry, University of Central Florida, 4000 Central Florida Boulevard, Orlando, FL 32816-2366, USA |
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Abstract: | Numerous large non-coding RNAs are rapidly being discovered, and many of them have been shown to play vital roles in gene expression, gene regulation, and human diseases. Given their often structured nature, specific recognition with an antibody fragment becomes feasible and may help define the structure and function of these non-coding RNAs. As demonstrated for protein antigens, specific antibodies may aid in RNA crystal structure elucidation or the development of diagnostic tools and therapeutic drugs targeting disease-causing RNAs. Recent success and limitation of RNA antibody development has made it imperative to generate an effective antibody library specifically targeting RNA molecules. Adopting the reduced chemical diversity design and further restricting the interface diversity to tyrosines, serines, glycines, and arginines only, we have constructed a RNA-targeting Fab library. Phage display selection and downstream characterization showed that this library yielded high-affinity Fabs for all three RNA targets tested. Using a quantitative specificity assay, we found that these Fabs are highly specific, possibly due to the alternate codon design we used to avoid consecutive arginines in the Fab interface. In addition, the effectiveness of the minimal Fab library may challenge our view of the protein–RNA binding interface and provide a unique solution for future design of RNA-binding proteins. |
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Keywords: | ncRNA, non-protein coding RNA lncRNA, long non-coding RNA CDR, complementarity-determining region ssDNA, single-stranded DNA EMSA, electrophoretic mobility shift assay PACE, polyacrylamide coelectrophoresis FSI, Fab specificity index EDTA, ethylenediaminetetraacetic acid PBS, phosphate-buffered saline BSA, bovine serum albumin |
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