| Structural and catalytic RNAs play central roles in fundamental cellular processes such as transcription, translation, control of gene expression, and embryonic development, and in many cases their three-dimensional structure is directly related to function. X-ray crystallography has been the method of choice for determining the high-resolution structures of large RNAs, but the preparation of diffraction-quality crystals remains a major bottleneck in RNA crystallography. We describe a general approach enabling the selection and affinity maturation of specific, high-affinity antibody fragments targeted against RNA. The key steps of this approach, phage display and error-prone PCR, are unbiased and can presumably be adopted for any RNA target of interest. Highly sequence specific, high-affinity Fabs were selected and affinity-matured against the class I ligase RNA. We have validated the use of Fabs as RNA crystallization chaperones by solving the crystal structure of the Fab-ligase complex at 3.1A resolution, and we envision the use of this approach for diverse RNA targets of biological importance which have previously eluded crystallographic analysis. |
