4XNR: Vibrio Vulnificus Adenine Riboswitch Aptamer Domain, Synthesized by Position-selective Labeling of RNA (PLOR), in Complex with Adenine

Citation:
Abstract
Knowledge of the structure and dynamics of RNA molecules is critical to understanding their many biological functions. Furthermore, synthetic RNAs have applications as therapeutics and molecular sensors. Both research and technological applications of RNA would be dramatically enhanced by methods that enable incorporation of modified or labelled nucleotides into specifically designated positions or regions of RNA. However, the synthesis of tens of milligrams of such RNAs using existing methods has been impossible. Here we develop a hybrid solid-liquid phase transcription method and automated robotic platform for the synthesis of RNAs with position-selective labelling. We demonstrate its use by successfully preparing various isotope- or fluorescently labelled versions of the 71-nucleotide aptamer domain of an adenine riboswitch for nuclear magnetic resonance spectroscopy or single-molecule Forster resonance energy transfer, respectively. Those RNAs include molecules that were selectively isotope-labelled in specific loops, linkers, a helix, several discrete positions, or a single internal position, as well as RNA molecules that were fluorescently labelled in and near kissing loops. These selectively labelled RNAs have the same fold as those transcribed using conventional methods, but they greatly simplify the interpretation of NMR spectra. The single-position isotope- and fluorescently labelled RNA samples reveal multiple conformational states of the adenine riboswitch. Lastly, we describe a robotic platform and the operation that automates this technology. Our selective labelling method may be useful for studying RNA structure and dynamics and for making RNA sensors for a variety of applications including cell-biological studies, substance detection, and disease diagnostics.
PDB ID: 4XNRDownload
MMDB ID: 129105
PDB Deposition Date: 2015/1/16
Updated in MMDB: 2017/12
Experimental Method:
x-ray diffraction
Resolution: 2.21  Å
Biological Unit for 4XNR: monomeric; determined by author
Molecular Components in 4XNR
Label Count Molecule
Nucleotide(1 molecule)
1
Vibrio Vulnificus Adenine Riboswitch
Molecule annotation
Chemicals (7 molecules)
1
1
2
6
* Click molecule labels to explore molecular sequence information.

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