Mapping RNA Structure In Vitro with SHAPE Chemistry and Next-Generation Sequencing (SHAPE-Seq)

  • Kyle E. Watters
  • Julius B. Lucks
Part of the Methods in Molecular Biology book series (MIMB, volume 1490)


Mapping RNA structure with selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry has proven to be a versatile method for characterizing RNA structure in a variety of contexts. SHAPE reagents covalently modify RNAs in a structure-dependent manner to create adducts at the 2′-OH group of the ribose backbone at nucleotides that are structurally flexible. The positions of these adducts are detected using reverse transcriptase (RT) primer extension, which stops one nucleotide before the modification, to create a pool of cDNAs whose lengths reflect the location of SHAPE modification. Quantification of the cDNA pools is used to estimate the “reactivity” of each nucleotide in an RNA molecule to the SHAPE reagent. High reactivities indicate nucleotides that are structurally flexible, while low reactivities indicate nucleotides that are inflexible. These SHAPE reactivities can then be used to infer RNA structures by restraining RNA structure prediction algorithms. Here, we provide a state-of-the-art protocol describing how to perform in vitro RNA structure probing with SHAPE chemistry using next-generation sequencing to quantify cDNA pools and estimate reactivities (SHAPE-Seq). The use of next-generation sequencing allows for higher throughput, more consistent data analysis, and multiplexing capabilities. The technique described herein, SHAPE-Seq v2.0, uses a universal reverse transcription priming site that is ligated to the RNA after SHAPE modification. The introduced priming site allows for the structural analysis of an RNA independent of its sequence.

Key words

SHAPE SHAPE-Seq RNA RNA structure probing RNA structure mapping Next-generation sequencing RNA structure RNA folding 



We thank Alex Settle for assistance on experimental procedures, Peter Schweitzer and the Cornell Life Sciences Core facility for sequencing support, and David Loughrey and James Chappell for helpful comments in reviewing this manuscript. This work was supported by the National Science Foundation Graduate Research Fellowship Program (grant number DGE-1144153 to K.E.W.); the Cornell University Center for Life Sciences Enterprises, a New York State Center for Advanced Technology supported by New York State and industrial partners (grant number C110124 to J.B.L.); and a New Innovator Award through the National Institute of General Medical Sciences of the National Institutes of Health (grant number DP2GM110838 to J.B.L.). K.E.W. is a Fleming Scholar in the Robert F. Smith School of Chemical and Biomolecular Engineering at Cornell University. J.B.L. is an Alfred P. Sloan Research Fellow.


  1. 1.
    Weeks KM (2010) Advances in RNA structure analysis by chemical probing. Curr Opin Struct Biol 20:295–304CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Butcher SE, Pyle AM (2011) The molecular interactions that stabilize RNA tertiary structure: RNA motifs, patterns, and networks. Acc Chem Res 44:1302–1311CrossRefPubMedGoogle Scholar
  3. 3.
    Duncan CDS, Weeks KM (2008) SHAPE analysis of long-range interactions reveals extensive and thermodynamically preferred misfolding in a fragile group I intron RNA. Biochemistry 47:8504–8513CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    McGinnis JL, Weeks KM (2014) Ribosome RNA assembly intermediates visualized in living cells. Biochemistry 53:3237–3247CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Rouskin S, Zubradt M, Washietl S et al (2014) Genome-wide probing of RNA structure reveals active unfolding of mRNA structures in vivo. Nature 505:701–705CrossRefPubMedGoogle Scholar
  6. 6.
    Ding Y, Tang Y, Kwok CK et al (2014) In vivo genome-wide profiling of RNA secondary structure reveals novel regulatory features. Nature 505:696–700CrossRefPubMedGoogle Scholar
  7. 7.
    Talkish J, May G, Lin Y et al (2014) Mod-seq: high-throughput sequencing for chemical probing of RNA structure. RNA 20:713–720CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Siegfried NA, Busan S, Rice GM et al (2014) RNA motif discovery by SHAPE and mutational profiling (SHAPE-MaP). Nat Methods 11:959–965CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Homan PJ, Tandon A, Rice GM et al (2014) RNA tertiary structure analysis by 2′-hydroxyl molecular interference. Biochemistry 53:6825–6833CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Cheng C, Chou FC, Kladwang W et al (2015) Consistent global structures of complex RNA states through multidimensional chemical mapping. eLife 4, e07600PubMedPubMedCentralGoogle Scholar
  11. 11.
    Byrne RT, Konevega AL, Rodnina MV, Antson AA (2010) The crystal structure of unmodified tRNAPhe from Escherichia coli. Nucleic Acids Res 38:4154–4162CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Loughrey D, Watters KE, Settle AH, Lucks JB (2014) SHAPE-Seq 2.0: systematic optimization and extension of high-throughput chemical probing of RNA secondary structure with next generation sequencing. Nucleic Acids Res 42:e165CrossRefPubMedCentralGoogle Scholar
  13. 13.
    Cordero P, Lucks JB, Das R (2012) An RNA mapping DataBase for curating RNA structure mapping experiments. Bioinformatics 28:3006–3008CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Peattie DA, Gilbert W (1980) Chemical probes for higher-order structure in RNA. Proc Natl Acad Sci U S A 77:4679–4682CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    McGinnis JL, Dunkle JA, Cate JHD, Weeks KM (2012) The mechanisms of RNA SHAPE chemistry. J Am Chem Soc 134:6617–6624CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Culver GM, Noller HF (1998) Directed hydroxyl radical probing of 16S ribosomal RNA in ribosomes containing Fe(II) tethered to ribosomal protein S20. RNA 4:1471–1480CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Brunel C, Romby P (2000) Probing RNA structure and RNA-ligand complexes with chemical probes. Methods Enzymol 318:3–21CrossRefPubMedGoogle Scholar
  18. 18.
    Mortimer SA, Weeks KM (2007) A fast-acting reagent for accurate analysis of RNA secondary and tertiary structure by SHAPE chemistry. J Am Chem Soc 129:4144–4145CrossRefPubMedGoogle Scholar
  19. 19.
    Spitale RC, Flynn RA, Torre EA et al (2014) RNA structural analysis by evolving SHAPE chemistry. Wiley Interdiscip Rev RNA 5:867–881CrossRefPubMedGoogle Scholar
  20. 20.
    Wilkinson KA, Merino EJ, Weeks KM (2006) Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE): quantitative RNA structure analysis at single nucleotide resolution. Nat Protoc 1:1610–1616CrossRefPubMedGoogle Scholar
  21. 21.
    Merino EJ, Wilkinson KA, Coughlan JL, Weeks KM (2005) RNA structure analysis at single nucleotide resolution by selective 2′-hydroxyl acylation and primer extension (SHAPE). J Am Chem Soc 127:4223–4231CrossRefPubMedGoogle Scholar
  22. 22.
    Aviran S, Trapnell C, Lucks JB et al (2011) Modeling and automation of sequencing-based characterization of RNA structure. Proc Natl Acad Sci U S A 108:11069–11074CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Aviran S, Lucks JB, Pachter L (2011) RNA structure characterization from chemical mapping experiments. 49th annual Allerton Conference on communication, control, and computing. pp 1743–1750. doi: 10.1109/Allerton.2011.6120379
  24. 24.
    Steen K-A, Rice GM, Weeks KM (2012) Fingerprinting noncanonical and tertiary RNA structures by differential SHAPE reactivity. J Am Chem Soc 134:13160–13163CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Mathews DH, Disney MD, Childs JL et al (2004) Incorporating chemical modification constraints into a dynamic programming algorithm for prediction of RNA secondary structure. Proc Natl Acad Sci U S A 101:7287–7292CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Low JT, Weeks KM (2010) SHAPE-directed RNA secondary structure prediction. Methods 52:150–158CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Vasa SM, Guex N, Wilkinson KA et al (2008) ShapeFinder: a software system for high-throughput quantitative analysis of nucleic acid reactivity information resolved by capillary electrophoresis. RNA 14:1979–1990CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Lucks JB, Mortimer SA, Trapnell C et al (2011) Multiplexed RNA structure characterization with selective 2′-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq). Proc Natl Acad Sci U S A 108:11063–11068CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Mortimer SA, Trapnell C, Aviran S et al (2012) SHAPE-Seq: high-throughput RNA structure analysis. Curr Protoc Chem Biol 4:275–297PubMedGoogle Scholar
  30. 30.
    Avis JM, Conn GL, Walker SC (2012) Cis-acting ribozymes for the production of RNA in vitro transcripts with defined 5′ and 3′ ends. Methods Mol Biol 941:83–98CrossRefPubMedGoogle Scholar
  31. 31.
    Kao C, Zheng M, Rüdisser S (1999) A simple and efficient method to reduce nontemplated nucleotide addition at the 3 terminus of RNAs transcribed by T7 RNA polymerase. RNA 5:1268–1272CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Turner R, Shefer K, Ares M (2013) Safer one-pot synthesis of the “SHAPE” reagent 1-methyl-7-nitroisatoic anhydride (1m7). RNA 19:1857–1863CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Mortimer SA, Weeks KM (2009) Time-resolved RNA SHAPE chemistry: quantitative RNA structure analysis in one-second snapshots and at single-nucleotide resolution. Nat Protoc 4:1413–1421CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Hajdin CE, Bellaousov S, Huggins W et al (2013) Accurate SHAPE-directed RNA secondary structure modeling, including pseudoknots. Proc Natl Acad Sci U S A 110:5498–5503CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Robert F. Smith School of Chemical and Biomolecular EngineeringCornell UniversityIthacaUSA

Personalised recommendations