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

Date Published: April 15, 2019

Publisher: Public Library of Science

Author(s): Jason R. Stagno, Ping Yu, Marzena A. Dyba, Yun-Xing Wang, Yu Liu, Petri Kursula.


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.

Partial Text

The ongoing discovery of numerous biologically important RNAs demands the determination of their structures to better understand their functions. X-ray crystallography contributes the majority of RNA three-dimensional structures in the PDB [1]. Solving the phase problem is the major bottleneck between X-ray diffraction data and refined structures. Obtaining phase information in the case of RNA is often challenging, and most often requires de novo phase determination using anomalous or isomorphous difference data from heavy-atom derivatives [2–5]. Heavy-atom derivatization strategies have been widely used to enable crystallographic phasing [1, 2, 6]. Soaking RNA crystals in buffers containing heavy atoms (e.g., halides, metal ions) offers a straightforward approach to introduce anomalous scatterers into the ordered solvent shell surrounding the RNA molecules. However, not all crystals can tolerate such soaking, which may result in crystal cracking, deterioration, and loss in diffraction quality. In addition, heavy-atom incorporation by soaking is not guaranteed, and the number and location of binding sites is often unpredictable. Engineered sequences, for example, a tandem G-U wobble pair cation binding motif, can be inserted into RNAs to improve the binding specificity of heavy-atoms [2]. Such strategies require mutations in some non-functional regions of an RNA molecule. Alternatively, RNA molecules can be derivatized with halogenated nucleotides by incorporating them during RNA synthesis. Modified RNAs are usually prepared by in vitro transcription or solid-phase chemical synthesis methods. In vitro transcription can produce RNAs with lengths varying from tens to thousands of nucleotides (nt), but it is usually applied for producing RNAs labeled specifically by nucleotide type rather than at selected positions. Solid-phase chemical synthesis is the most widely used method for preparing site-specifically labeled RNAs. However, heavy-atom labeled phosphoramidites are not always commercially available, and it is challenging to incorporate with adequate efficiency heavy atoms at specific positions of RNAs larger than 60 nt in length using step-wise chemical synthesis [7]. This size limitation can be alleviated by enzymatically ligating short synthetic RNAs fragments [8]. However, discrete and complicated labeling schemes may be difficult to achieve by ligation due to low efficiencies and limited site choices.

The process for obtaining heavy-atom modified RNAs is a key bottleneck in solving unique biologically important RNA structures by X-ray crystallography. The introduction of heavy-atoms into RNA can be routinely achieved by PLOR, with the aim of breaking the phase ambiguity of crystallographic data. Such an application for PLOR is especially feasible for large RNAs that cannot be synthesized by solid-phase synthesis with high-enough efficiency. The easy scale-up of PLOR also makes it practical to carry out initial crystallization screening with heavy-atom containing RNAs. This avoids the potential pitfalls of non-isomorphism between native and derivative crystals. Covalent incorporation of heavy atoms into RNA at desired positions using PLOR offers a general strategy for SAD/MAD phasing of novel RNA crystal structures. This application of PLOR can be extended to structure determination of RNA/protein complexes by derivatizing RNA instead of, or in addition to, the protein, thereby increasing the chances for successful de novo phasing. It is straightforward to extend the strategy to synthesize RNAs containing other heavy-atoms, including Cl, Br, Se, etc. by PLOR. This application of PLOR will hasten the rate of novel RNA structure determination by X-ray crystallography.