Use Dimethyl Sulphate Methylation of Unprotected Adenines and Cytosines

Structure-Seq profiles RNA structures for in vivo or in vitro applications with single-nucleotide resolution (Ding et al., 2014) (Ding et al., 2015). This method identifies secondary RNA structures by using the chemical modification induced by DMS on unpaired adenines and cytosines.Briefly, samples are treated with DMS to mark secondary RNA structures in vivo. The RNA is extracted, poly(A)-selected, and treated with DNase. Using random hexamers as primers, reverse transcription is initiated. The resulting single-stranded DNA is ligated to single-stranded DNA linkers and self-circularized using CircLigase enzyme. Next, the DNA is PCR-amplified, size-selected, and sequenced.

Similar methods: icSHAPE, Mod-Seq, DMS-seq, PARS, Frag-seq, dsRNA-Seq


  • Provides genome-wide profiling of RNA structures at single-nucleotide resolution
  • Can be used for in vivo and in vitro applications
  • DMS is cell-permeable and can be used for in vivo applications
  • One RT primer synthesis retrieves information for tens of thousands of RNA structures
  • Random-hexamer primers minimize 3′ end bias


  • RBPs can block DMS modification in vivo
  • Circularization may introduce additional bias


Illumina Library prep and Array Kit Selector


Fang Y. and Fullwood M. J. Roles, Functions, and Mechanisms of Long Non-coding RNAs in Cancer. Genomics Proteomics Bioinformatics. 2016;14:42-54

Reuter J. A., Spacek D. V. and Snyder M. P. High-Throughput Sequencing Technologies. Mol Cell. 2015;58:586-597


Fang R., Moss W. N., Rutenberg-Schoenberg M. and Simon M. D. Probing Xist RNA Structure in Cells Using Targeted Structure-Seq. PLoS Genet. 2015;11:e1005668