RNA Structure Analysis by Chemical Probing with DMS and CMCT
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RNA structure is important for understanding RNA function and stability within a cell. Chemical probing is a well-established and convenient method to evaluate the structure of an RNA. Several structure-sensitive chemicals can differentiate paired and unpaired nucleotides. This chapter specifically addresses the use of DMS and CMCT. Although exhibiting different affinities, the combination of these two chemical reagents enables screening of all four nucleobases. DMS and CMCT are only reactive with exposed unpaired nucleotides. We have used this method to analyze the effect of the RNA chaperone Hfq on the conformation of the 16S rRNA. The strategy here described may be applied for the study of many other RNA-binding proteins and RNAs.
Key wordsChemical probing CMCT DMS Primer extension rRNA RNA fold RNA secondary structure
This work was financially supported by Project LISBOA-01-0145-FEDER-007660 (Microbiologia Molecular, Estrutural e Celular) funded by FEDER through COMPETE2020-Programa Operacional Competitividade e Internacionalização (POCI) and by FCT-Fundação para a Ciência e a Tecnologia (Portugal), including Program IF (IF/00961/2014) and Grants PTDC/BIA-MIC/32525/2017 to J.M.A. and PTDC/BIA-MIC/1399/2014 to CMA; R.F.dS. is recipient of an FCT Doctoral fellowship (PD/BD/105733/2014). We also acknowledge the European Union Horizon 2020 Research and Innovation Programme grant agreement no. 635536 to CMA.
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