Heavy-atom labeling of RNA by PLOR for de novo crystallographic phasing

• Published in: PloS one 2019-04, Vol.14 (4), p.e0215555
• Main Authors: Stagno, Jason R, Yu, Ping, Dyba, Marzena A, Wang, Yun-Xing, Liu, Yu
• Format: Article
• Language: English
• Subjects: Acids; Adenine; Aptamers; Atoms; BASIC BIOLOGICAL SCIENCES; Binding sites; Biology and life sciences; Biophysics; Cancer; Chemical synthesis; Composition; Crystal structure; Crystal structure refinement; Crystallization; Crystallography; Crystallography, X-Ray; Crystals; Identification and classification; Iodine; Labeling; Labelling; Laboratories; Medical research; Methods; Models, Molecular; Molecular modeling; Nucleotides; Physical Sciences; Proteins; Purines; Research and analysis methods; Ribonucleic acid; Riboswitch; RNA; RNA polymerase; RNA structure; RNA synthesis; RNA transcription labeling; Staining and Labeling; X-ray crystallography
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0215555

Due to the paucity of known RNA structures, experimental phasing is crucial for obtaining three-dimensional structures of RNAs by X-ray crystallography. Covalent attachment of heavy atoms to RNAs is one of the most useful strategies to facilitate phase determination. However, this approach is limited by the inefficiency or inability to synthesize large RNAs (>60 nucleotides) site-specifically labeled with heavy atoms using traditional methods. Here, we applied our recently reported method, PLOR (position-selective labeling of RNA) to incorporate 5-iodouridine at specific positions in the adenine riboswitch RNA aptamer domain, which was then used for crystallization and subsequent de novo SAD phasing. PLOR is a powerful tool to improve the efficiency of obtaining RNA structures de novo by X-ray crystallography.