Advertisement

RNA Chaperones pp 209-223 | Cite as

RNA Structure Analysis by Chemical Probing with DMS and CMCT

  • José M. AndradeEmail author
  • Ricardo F. dos Santos
  • Cecília M. ArraianoEmail author
Protocol
  • 291 Downloads
Part of the Methods in Molecular Biology book series (MIMB, volume 2106)

Abstract

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 words

Chemical probing CMCT DMS Primer extension rRNA RNA fold RNA secondary structure 

Notes

Acknowledgments

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.

References

  1. 1.
    dos Santos RF, Quendera AP, Boavida S et al (2018) Major 3′–5′ exoribonucleases in the metabolism of coding and non-coding RNA. In: Teplow DB (ed) Progress in molecular biology and translational science. Academic Press, Cambridge, pp 101–155Google Scholar
  2. 2.
    Weeks KM (2010) Advances in RNA structure analysis by chemical probing. Curr Opin Struct Biol 20:295–304CrossRefGoogle Scholar
  3. 3.
    Andrade JM, Pobre V, Arraiano CM (2013) Small RNA modules confer different stabilities and interact differently with multiple targets. PLoS One 8:e52866CrossRefGoogle Scholar
  4. 4.
    Brunel C, Romby P (2000) Probing RNA structure and RNA-ligand complexes with chemical probes. Methods Enzymol 318:3–21CrossRefGoogle Scholar
  5. 5.
    Ehresmann C, Baudin F, Mougel M et al (1987) Probing the structure of RNAs in solution. Nucleic Acids Res 15:9109–9128CrossRefGoogle Scholar
  6. 6.
    Marryman C, Noller HF (1998) Footprinting and modification-interference analysis of bindind sites on RNA. In: Smith CWJ (ed) RNA-protein interactions: a practical approach. Oxford University Press, Oxford, pp 237–254Google Scholar
  7. 7.
    Ziehler WA, Engelke DR (2001) Probing RNA structure with chemical reagents and enzymes. Curr Protoc Nucleic Acid Chem Chapter 6:Unit 6.1Google Scholar
  8. 8.
    Sachsenmaier N, Handl S, Debeljak F et al (2014) Mapping RNA structure in vitro using nucleobase-specific probes. Methods Mol Biol 1086:79–94CrossRefGoogle Scholar
  9. 9.
    Philippe J-V, Ayadi L, Branlant C et al (2015) Probing small non-coding RNAs structures. In: Rederstorff M (ed) Small non-coding RNAs. Methods in molecular biology. Humana Press, Totowa, pp 119–136CrossRefGoogle Scholar
  10. 10.
    Behm-Ansmant I, Helm M, Motorin Y (2011) Use of specific chemical reagents for detection of modified nucleotides in RNA. J Nucleic Acids 2011:1–17CrossRefGoogle Scholar
  11. 11.
    Tijerina P, Mohr S, Russell R (2007) DMS footprinting of structured RNAs and RNA–protein complexes. Nat Protoc 2:2608–2623CrossRefGoogle Scholar
  12. 12.
    Caprara M (2011) RNA structure determination using chemical and nuclease digestion methods. In: Rio D, Hannon G, Ares M et al (eds) RNA: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp 269–275Google Scholar
  13. 13.
    Yu AM, Evans ME, Lucks JB (2018) Estimating RNA structure chemical probing reactivities from reverse transcriptase stops and mutations. bioRXiv.  https://doi.org/10.1101/292532
  14. 14.
    Andrade JM, Dos Santos RF, Chelysheva I et al (2018) The RNA-binding protein Hfq is important for ribosome biogenesis and affects translation fidelity. EMBO J 37:e97631CrossRefGoogle Scholar
  15. 15.
    Wan Y, Kertesz M, Spitale RC et al (2011) Understanding the transcriptome through RNA structure. Nat Rev Genet 12:641–655CrossRefGoogle Scholar
  16. 16.
    Das R, Laederach A, Pearlman SM et al (2005) SAFA: semi-automated footprinting analysis software for high-throughput quantification of nucleic acid footprinting experiments. RNA 11:344–354CrossRefGoogle Scholar
  17. 17.
    Andrade JM, Pobre V, Matos AM et al (2012) The crucial role of PNPase in the degradation of small RNAs that are not associated with Hfq. RNA 18:844–855CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Instituto de Tecnologia Química e Biológica António XavierUniversidade Nova de LisboaOeirasPortugal

Personalised recommendations