Summary information and primary citation

PDB-id
5uza; DSSR-derived features in text and JSON formats
Class
RNA
Method
X-ray (2.22 Å)
Summary
Adenine riboswitch aptamer domain labelled with iodo-uridine by position-selective labelling of RNA (plor)
Reference
Liu Y, Holmstrom E, Yu P, Tan K, Zuo X, Nesbitt DJ, Sousa R, Stagno JR, Wang YX (2018): "Incorporation of isotopic, fluorescent, and heavy-atom-modified nucleotides into RNAs by position-selective labeling of RNA." Nat Protoc, 13, 987-1005. doi: 10.1038/nprot.2018.002.
Abstract
Site-specific incorporation of labeled nucleotides is an extremely useful synthetic tool for many structural studies (e.g., NMR, electron paramagnetic resonance (EPR), fluorescence resonance energy transfer (FRET), and X-ray crystallography) of RNA. However, specific-position-labeled RNAs >60 nt are not commercially available on a milligram scale. Position-selective labeling of RNA (PLOR) has been applied to prepare large RNAs labeled at desired positions, and all the required reagents are commercially available. Here, we present a step-by-step protocol for the solid-liquid hybrid phase method PLOR to synthesize 71-nt RNA samples with three different modification applications, containing (i) a 13C15N-labeled segment; (ii) discrete residues modified with Cy3, Cy5, or biotin; or (iii) two iodo-U residues. The flexible procedure enables a wide range of downstream biophysical analyses using precisely localized functionalized nucleotides. All three RNAs were obtained in <2 d, excluding time for preparing reagents and optimizing experimental conditions. With optimization, the protocol can be applied to other RNAs with various labeling schemes, such as ligation of segmentally labeled fragments.

Cartoon-block schematics in six views (download the tarball)

PyMOL session file Download PDB file View in 3Dmol.js