In Vitro Synthesis and RNA Structure Probing of CUG Triplet Repeat RNA

  • Remco T. P. van Cruchten
  • Derick G. WansinkEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2056)


Aberrant RNA structure plays a central role in the molecular mechanisms guided by repeat RNAs in diseases like myotonic dystrophy (DM), C9orf72-linked amyotrophic lateral sclerosis (ALS) and fragile X tremor/ataxia syndrome (FXTAS). Much knowledge remains to be gained about how these repeat-expanded transcripts are actually folded, especially regarding the properties specific to very long and interrupted repeats. RNA structure can be interrogated by chemical as well as enzymatic probes. These probes usually bind or cleave single-stranded nucleotides, which can subsequently be detected using reverse transcriptase primer extension. In this chapter, we describe methodology for in vitro synthesis and structure probing of expanded CUG repeat RNAs associated with DM type 1 and approaches for the associated data analysis.


CUG repeat DMPK In vitro transcription Myotonic dystrophy RNA folding RNA structure Hairpin SHAPE RNase T1 DMS 



We wish to thank Prof. Bé Wieringa for his contribution to supervision of this study as part of R.T.P. van Cruchten’s Ph.D. project and for critical reading of the manuscript.


  1. 1.
    Sobczak K, de Mezer M, Michlewski G, Krol J, Krzyzosiak WJ (2003) RNA structure of trinucleotide repeats associated with human neurological diseases. Nucleic Acids Res 31:5469–5482PubMedPubMedCentralCrossRefGoogle Scholar
  2. 2.
    Ciesiolka A, Jazurek M, Drazkowska K, Krzyzosiak WJ (2017) Structural characteristics of simple RNA repeats associated with disease and their deleterious protein interactions. Front Cell Neurosci 11:97PubMedPubMedCentralCrossRefGoogle Scholar
  3. 3.
    Zu T, Gibbens B, Doty NS, Gomes-pereira M, Huguet A, Stone MD (2010) Non-ATG – initiated translation directed by microsatellite expansions. Proc Natl Acad Sci U S A 108:260–265PubMedPubMedCentralCrossRefGoogle Scholar
  4. 4.
    Aartsma-Rus A, van Vliet L, Hirschi M, Janson AAM, Heemskerk H, de Winter CL et al (2009) Guidelines for antisense oligonucleotide design and insight into splice-modulating mechanisms. Mol Ther 17:548–553PubMedCrossRefGoogle Scholar
  5. 5.
    Bernat V, Disney MD (2015) RNA structures as mediators of neurological diseases and as drug targets. Neuron 87:28–46PubMedPubMedCentralCrossRefGoogle Scholar
  6. 6.
    Weeks KM (2010) Advances in RNA structure analysis by chemical probing. Curr Opin Struct Biol 20:295–304PubMedPubMedCentralCrossRefGoogle Scholar
  7. 7.
    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 127:4223–4231PubMedCrossRefGoogle Scholar
  8. 8.
    Lorenz R, Wolfinger MT, Tanzer A, Hofacker IL (2016) Predicting RNA secondary structures from sequence and probing data. Methods 103:86–98PubMedCrossRefGoogle Scholar
  9. 9.
    Spitale RC, Crisalli P, Flynn RA, Torre EA, Kool ET, Chang HY (2013) RNA SHAPE analysis in living cells. Nat Chem Biol 9:18–20PubMedCrossRefGoogle Scholar
  10. 10.
    Leeflang EP, Arnheim N (1995) A novel repeat structure at the myotonic dystrophy locus in a 37 repeat allele with unexpectedly high stability. Hum Mol Genet 4:135–136PubMedCrossRefGoogle Scholar
  11. 11.
    Busan S, Weeks KM (2013) Role of context in RNA structure: flanking sequences reconfigure CAG motif folding in huntingtin exon 1 transcripts. Biochemistry 52:8219–8225PubMedPubMedCentralCrossRefGoogle Scholar
  12. 12.
    van Cruchten RTP, Wieringa B, Wansink DG (2019) Expanded CUG repeats in DMPK transcripts adopt diverse hairpin conformations without influencing the structure of the flanking sequences. RNA 25:481–495PubMedPubMedCentralCrossRefGoogle Scholar
  13. 13.
    Figura G, Koscianska E, Krzyzosiak WJ (2015) In vitro expansion of CAG, CAA, and mixed CAG/CAA repeats. Int J Mol Sci 16:18741–18751PubMedPubMedCentralCrossRefGoogle Scholar
  14. 14.
    Napierała M, Krzyzosiak WJ (1997) CUG repeats present in myotonin kinase RNA form metastable “slippery” hairpins. J Biol Chem 272:31079–31085PubMedCrossRefGoogle Scholar
  15. 15.
    Mitra S, Shcherbakova IV, Altman RB, Brenowitz M, Laederach A (2008) High-throughput single-nucleotide structural mapping by capillary automated footprinting analysis. Nucleic Acids Res 36:1–10CrossRefGoogle Scholar
  16. 16.
    Cantara WA, Hatterschide J, Wu W, Musier-Forsyth K (2017) RiboCAT: a new capillary electrophoresis data analysis tool for nucleic acid probing. RNA 23:240–249PubMedPubMedCentralCrossRefGoogle Scholar
  17. 17.
    Kim H, Cordero P, Das R, Yoon S (2013) HiTRACE-web: an online tool for robust analysis of high-throughput capillary electrophoresis. Nucleic Acids Res 41:492–498CrossRefGoogle Scholar
  18. 18.
    Vasa SM, Guex N, Wilkinson KA, Weeks KM, Giddings MC (2008) ShapeFinder: a software system for high-throughput quantitative analysis of nucleic acid reactivity information resolved by capillary electrophoresis. RNA 14:1979–1990PubMedPubMedCentralCrossRefGoogle Scholar
  19. 19.
    Karabiber F, McGinnis JL, Favorov OV, Weeks KM (2013) QuShape: rapid, accurate, and best-practices quantification of nucleic acid probing information, resolved by capillary electrophoresis. RNA 19:63–73PubMedPubMedCentralCrossRefGoogle Scholar
  20. 20.
    Reuter JS, Mathews DH (2010) RNAstructure: software for RNA secondary structure prediction and analysis. BMC Bioinformatics 11:129PubMedPubMedCentralCrossRefGoogle Scholar
  21. 21.
    Lorenz R, Bernhart SH, Höner zu Siederdissen C, Tafer H, Flamm C, Stadler PF et al (2011) ViennaRNA package 2.0. Algorithms Mol Biol 6:26PubMedPubMedCentralCrossRefGoogle Scholar
  22. 22.
    Orpana AK, Ho TH, Alagrund K, Ridanpää M, Aittomäki K, Stenman J (2013) Novel heat pulse extension-PCR-based method for detection of large CTG-repeat expansions in myotonic dystrophy type 1. J Mol Diagn 15:110–115PubMedCrossRefGoogle Scholar
  23. 23.
    Meng YX, Shen HR, Zhao Z, Bing Q, Li N, Hu J (2015) Optimization PCR for detection CTG/CCTG-repeat expansions in the diagnosis of myotonic dystrophies. Ann Clin Lab Sci 45:502–507PubMedGoogle Scholar
  24. 24.
    Draper DE (2004) A guide to ions and RNA structure. RNA 10:335–343PubMedPubMedCentralCrossRefGoogle Scholar
  25. 25.
    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 1:1610–1616PubMedCrossRefGoogle Scholar
  26. 26.
    Carrell ST, Tang Z, Mohr S, Lambowitz AM, Thornton CA (2018) Detection of expanded RNA repeats using thermostable group II intron reverse transcriptase. Nucleic Acids Res 46:e1PubMedCrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Remco T. P. van Cruchten
    • 1
  • Derick G. Wansink
    • 1
    Email author
  1. 1.Department of Cell Biology, Radboud Institute for Molecular Life SciencesRadboud University Medical CenterNijmegenThe Netherlands

Personalised recommendations