Publication Date:
2015-05-06
Description:
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.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Liu, Yu -- Holmstrom, Erik -- Zhang, Jinwei -- Yu, Ping -- Wang, Jinbu -- Dyba, Marzena A -- Chen, De -- Ying, Jinfa -- Lockett, Stephen -- Nesbitt, David J -- Ferre-D'Amare, Adrian R -- Sousa, Rui -- Stagno, Jason R -- Wang, Yun-Xing -- HHSN261200800001E/PHS HHS/ -- R01 GM052522/GM/NIGMS NIH HHS/ -- T32 GM-065103/GM/NIGMS NIH HHS/ -- Intramural NIH HHS/ -- England -- Nature. 2015 Jun 18;522(7556):368-72. doi: 10.1038/nature14352. Epub 2015 May 4.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Protein-Nucleic Acid Interaction Section, Structural Biophysics Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, Maryland 21702, USA. ; JILA, National Institute of Standards and Technology and Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309, USA. ; Biochemistry and Biophysics Center, National Heart, Lung and Blood Institute, Bethesda, Maryland 20892, USA. ; Structural Biophysics Laboratory, Basic Science Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702, USA. ; Optical Microscopy and Analysis Laboratory, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702, USA. ; Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA. ; Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/25938715" target="_blank"〉PubMed〈/a〉
Keywords:
Adenine/analysis/chemistry/metabolism
;
Aptamers, Nucleotide/analysis/chemistry/metabolism
;
Automation/methods
;
Base Sequence
;
Biosensing Techniques
;
DNA/genetics/metabolism
;
*Fluorescence
;
Fluorescence Resonance Energy Transfer
;
In Vitro Techniques
;
Isotope Labeling/*methods
;
Magnetic Resonance Spectroscopy
;
Molecular Sequence Data
;
Nucleic Acid Conformation
;
RNA/analysis/*chemical synthesis/*chemistry/genetics
;
Riboswitch/genetics
;
Robotics
;
Templates, Genetic
;
Transcription, Genetic
Print ISSN:
0028-0836
Electronic ISSN:
1476-4687
Topics:
Biology
,
Chemistry and Pharmacology
,
Medicine
,
Natural Sciences in General
,
Physics
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