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Article Dans Une Revue Methods in Molecular Biology Année : 2021

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

Fatima Alghoul
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Gilbert Eriani
Franck Martin

Résumé

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).
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Dates et versions

hal-03194968 , version 1 (09-04-2021)

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Fatima Alghoul, Gilbert Eriani, Franck Martin. RNA Secondary Structure Study by Chemical Probing Methods Using DMS and CMCT.. Methods in Molecular Biology, 2021, Small Non-Coding RNAs, 2300, pp.241-250. ⟨10.1007/978-1-0716-1386-3_18⟩. ⟨hal-03194968⟩
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