Incorporation of isotopic, fluorescent, and heavy-atom-modified nucleotides into RNAs by position-selective labeling of RNA
- Shanghai Tong Univ., Shanghai (China); National Institutes of Health, Frederick, MD (United States)
- Univ. of Colorado, Boulder, CO (United States); Univ. of Zurich, Zurich (Switzerland)
- National Institutes of Health, Frederick, MD (United States)
- Argonne National Lab. (ANL), Argonne, IL (United States)
- Univ. of Colorado, Boulder, CO (United States)
- Univ. of Texas Health Science Center, San Antonio, TX (United States)
Here, 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 (CN)-C-13-N-15-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.
- Research Organization:
- Argonne National Laboratory (ANL), Argonne, IL (United States)
- Sponsoring Organization:
- National Institutes of Health (NIH), National Cancer Institute; National Science Foundation (NSF); National Institute of Standards and Technology (NIST); USDOE
- Grant/Contract Number:
- AC02-06CH11357
- OSTI ID:
- 1461437
- Journal Information:
- Nature Protocols, Vol. 13, Issue 5; ISSN 1754-2189
- Publisher:
- Nature Publishing GroupCopyright Statement
- Country of Publication:
- United States
- Language:
- English
Web of Science
Efficient labeling of organic molecules using 13 C elemental carbon: universal access to 13 C 2 -labeled synthetic building blocks, polymers and pharmaceuticals
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journal | January 2020 |
Strategic labelling approaches for RNA single-molecule spectroscopy
|
journal | April 2019 |
Heavy-atom labeling of RNA by PLOR for de novo crystallographic phasing
|
journal | April 2019 |
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