Chemical Inference of RNA Structures

CIRS-Seq was developed to investigate the complexity of secondary RNA structures in the mammalian transcriptome (Incarnato et al., 2014). CIRS-Seq uses DMS to methylate the N1 of adenosine and N3 of cytosine residues, and CMC to modify pseudouridines selectively, but only when they are in single-stranded conformation. Modifications on these nucleotide residues halt the RT process, effectively producing a truncated cDNA as a marker for the location of secondary RNA structures.

Briefly, cells are lysed and treated with proteinase K to dissociate protein-bound RNAs while leaving RNA secondary structures intact. The lysates are separated into 3 different treatment linesãDMS, CMC, and no treatment. In all 3 treatment lines, total RNA is extracted and reverse-transcribed using random primers. The resulting cDNA is isolated, ligated to sequencing adapters, and subjected to high-throughput sequencing. Reads from the DMS and CMC lines are used to identify the locations of secondary RNA structures, while the control is used to reduce background noise.


  • Accurately predicts secondary RNA structures, and reveals features of mRNAs and ncRNAs
  • Provides single-base resolution
  • Can identify structural requirements for RBPs


  • CMC and DMS may react with non_secondary RNA structures


Illumina Library prep and Array Kit Selector


Zucchelli S., Patrucco L., Persichetti F., Gustincich S. and Cotella D. Engineering Translation in Mammalian Cell Factories to Increase Protein Yield: The Unexpected Use of Long Non-Coding SINEUP RNAs. Comput Struct Biotechnol J. 2016;14:404-410

Kwok C. K., Tang Y., Assmann S. M. and Bevilacqua P. C. The RNA structurome: transcriptome-wide structure probing with next-generation sequencing. Trends Biochem Sci. 2015;40:221-232

Strobel E. J., Watters K. E., Loughrey D. and Lucks J. B. RNA systems biology: uniting functional discoveries and structural tools to understand global roles of RNAs. Curr Opin Biotechnol. 2016;39:182-191


Incarnato D., Anselmi F., Morandi E., et al. High-throughput single-base resolution mapping of RNA 2′-O-methylated residues. Nucleic Acids Res. 2016;