Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021:2300:241-250.
doi: 10.1007/978-1-0716-1386-3_18.

RNA Secondary Structure Study by Chemical Probing Methods Using DMS and CMCT

Fatima Alghoul  1 , Gilbert Eriani  1 , Franck Martin  2
Affiliations

RNA Secondary Structure Study by Chemical Probing Methods Using DMS and CMCT

Fatima Alghoul et al. Methods Mol Biol. 2021.

Abstract

RNA folds into secondary structures that can serve in understanding various RNA functions (Weeks KM. Curr Opin Struct Biol 20(3):295-304, 2010). Chemical probing is a method that enables the characterization of RNA secondary structures using chemical reagents that specifically modify RNA nucleotides that are located in single-stranded areas. In our protocol, we used Dimethyl Sulfate (DMS) and Cyclohexyl-3-(2-Morpholinoethyl) Carbodiimide metho-p-Toluene sulfonate (CMCT) that are both base-specific modifying reagents (Behm-Ansmant I, et al. J Nucleic Acids 2011:408053, 2011). These modifications are mapped by primer extension arrests using 5' fluorescently labeled primers. In this protocol, we show a comprehensive method to identify RNA secondary structures in vitro using fluorescently labeled oligos. To demonstrate the efficiency of the method, we give an example of a structure we have designed which corresponds to a part of the 5'-UTR regulatory element called Translation Inhibitory Element (TIE) from Hox a3 mRNA (Xue S, et al. Nature 517(7532):33-38, 2015).

Keywords: CMCT; Capillary electrophoresis; Chemical probing; DMS; Primer extension; QuSHAPE; RNA secondary structure.

PubMed Disclaimer

References

    1. Weeks KM (2010) Advances in RNA structure analysis by chemical probing. Curr Opin Struct Biol 20(3):295–304 - DOI
    1. Behm-Ansmant I, Helm M, Motorin Y (2011) Use of specific chemical reagents for detection of modified nucleotides in RNA. J Nucleic Acids 2011:408053 - DOI
    1. Xue S, Tian S, Fujii K, Kladwang W, Das R, Barna M (2015) RNA regulons in Hox 5' UTRs confer ribosome specificity to gene regulation. Nature 517(7532):33–38 - DOI
    1. Pestova TV, Kolupaeva VG (2002) The roles of individual eukaryotic translation initiation factors in ribosomal scanning and initiation codon selection. Genes Dev 16(22):2906–2922 - DOI
    1. Karabiber F, McGinnis JL, Favorov OV, Weeks KM (2013) QuShape: rapid, accurate, and best-practices quantification of nucleic acid probing information, resolved by capillary electrophoresis. RNA 19(1):63–73 - DOI

Publication types

LinkOut - more resources