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RNA Secondary Structure Prediction Using High-throughput SHAPE

Published: May 31st, 2013



1RT Biochemistry Section, HIV Drug Resistance Program, Frederick National Laboratory for Cancer Research
* These authors contributed equally

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

To understand the functions of catalytic and non-coding RNAs involved in regulation of splicing, translation, virus replication and cancer, a detailed knowledge of RNA structure is required1,2. Unfortunately, accurate prediction of RNA folding presents a formidable challenge. Classical probing agents suffer from many disadvantages such as toxicity, incomplete nucleotide coverage and/or throughput limited to 100-150 nucleotides per experiment. Unaided secondary structure prediction algorithms are similarly disadvantageous, owing to inaccuracies resulting from their inability to effectively distinguish among energetically similar structures. Large RNAs in par....

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Primer design and extension of the RNA 3' terminus

To analyze long RNAs by high-throughput SHAPE, a series of primer hybridization sites should be selected such that they (i) are separated by ~300 nt, (ii) are 20-30 nt in length, and (iii) that RNA/DNA hybrids produced by annealing DNA to these sites have an expected melting temperature of >50 °C. In addition, segments of RNA that are predicted to be highly structured should be avoided, although making such a determination requires some fo.......

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RNA containing the HIV-1 rev response element (RRE) and a 3' terminal structure cassette4 was prepared from a linearized plasmid by in vitro transcription, after which it was folded by heating, cooling, and incubation at 37 °C in the presence of MgCl2. RNA was exposed to NMIA and then reverse transcribed from a 5'-end-labeled DNA primer hybridized to the 3' terminal structure cassette. The resulting SHAPE cDNA library, together with control and sequencing reactions, was then fractionate.......

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We present here a detailed protocol for high-throughput SHAPE, a technique that allows secondary structure determination to single-nucleotide resolution for RNAs of any size. Moreover, coupling experimental SHAPE data with secondary structure prediction algorithms facilitates generation of RNA 2D models with a higher degree of accuracy than is possible with either method alone. The combination of fluorescently-labeled primers and automated CE provides significant advantages over the traditional gel-based SHAPE, facilitat.......

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S. Lusvarghi, J. Sztuba-Solinska, K.J. Purzycka, J.W. Rausch and S.F.J. Le Grice are supported by the Intramural Research Program of the National Cancer Institute, National Institutes of Health, USA.


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Name Company Catalog Number Comments
N-methylisatoic anhydride (NMIA) Life technologies M25 Dissolve in anhydrous DMSO
1-methyl-t-nitroisatoic anhydride (1M7) see ref. 22    
Superscript III Reverse Transcriptase Life technologies 18080044 10,000 units
Thermo sequenase cycle sequencing kit Affymetrix 78500  
      Materials provided by the user
RNA of interest     6 pmol per reaction (the limit of detection will be determined by the instrument)
Sets of four 5' labeled primers (Cy5, Cy5.5, WellRed D2 and WellRed D1/Licor IR800)     Primers are complementary to the RNA and are used in reverse transcription and sequencing reactions. The listed fluorophores are optimal for the Beckman Coulter 8000 CEQ. Primers may be purchased or synthesized in house.
DNA template     DNA is used for sequencing reactions, and must contain the sequence of the RNA being studied - including any 3'terminal extension, if present. Where applicable, it is often convenient to use the RNA transcription template.
10x RNA renaturation buffer     100 mM Tris-HCl pH 8.0, 1 M KCl, 1 mM EDTA
5X RNA folding buffer     200 mM Tris-HCl pH 8.0, 25 mM MgCl2, 2.5 mM EDTA, 650 mM KCl. (This buffer might be changed depending on the case (e.g. pH, EDTA, Mg, RNase inhibitor)
2.5X RT mix     4 μl 5X buffer, 1 μl 100 mM DTT, 1.5 μl water,1 μl 10 mM dNTPs, 0.5 μl SuperScript III. Note that the 5X buffer and 100 mM DTT are provided with purchase of SuperScript III (Invitrogen).
GenomeLab Sample Loading Solution (Beckman Coulter)     Attention: Avoid multiple freeze-thaw cycles
Capillary electrophoresis Beckman CEQ8000  
Thermocycler varies    

  1. Scott, W. G., Martick, M., Chi, Y. I. Structure and function of regulatory RNA elements: ribozymes that regulate gene expression. Biochim. Biophys. Acta. 1789, 634-641 (2009).
  2. Moore, P. B., Steitz, T. A. The roles of RNA in the synthesis of protein. Cold Spring Harb. Perspect. Biol. 3, a003780 (2011).
  3. Wilkinson, K. A., et al. High-throughput SHAPE analysis reveals structures in HIV-1 genomic RNA strongly conserved across distinct biological states. Plos Biol. 6, 883-899 (2008).
  4. Merino, E. J., Wilkinson, K. A., Coughlan, J. L., Weeks, K. M. RNA structure analysis at single nucleotide resolution by selective 2 '-hydroxyl acylation and primer extension (SHAPE). J. Am. Chem. Soc. 127, 4223-4231 (2005).
  5. Watts, J. M., et al. Architecture and secondary structure of an entire HIV-1 RNA genome. Nature. 460, 711-716 (2009).
  6. Xu, W., Bolduc, F., Hong, N., Perreault, J. P. The use of a combination of computer-assisted structure prediction and SHAPE probing to elucidate the secondary structures of five viroids. Mol. Plant Pathol. , (2012).
  7. Novikova, I. V., Hennelly, S. P., Sanbonmatsu, K. Y. Structural architecture of the human long non-coding RNA, steroid receptor RNA activator. Nucleic Acids Res. 40, 5034-5051 (2012).
  8. Leshin, J. A., Heselpoth, R., Belew, A. T., Dinman, J. High-throughput structural analysis of yeast ribosomes using hSHAPE. RNA Biol. 8, 478-487 (2011).
  9. Souliere, M. F., Haller, A., Rieder, R., Micura, R. A powerful approach for the selection of 2-aminopurine substitution sites to investigate RNA folding. J. Am. Chem. Soc. 133, 16161-16167 (2011).
  10. Wilkinson, K. A., Merino, E. J., Weeks, K. M. Selective 2 '-hydroxyl acylation analyzed by primer extension (SHAPE): quantitative RNA structure analysis at single nucleotide resolution. Nat. Protoc. 1, 1610-1616 (2006).
  11. McGinnis, J. L., Duncan, C. D. S., Weeks, K. M. High-Throughput Shape and Hydroxyl Radical Analysis of Rna Structure and Ribonucleoprotein Assembly. Method Enzymol. 468, 67-89 (2009).
  12. Low, J. T., Weeks, K. M. SHAPE-directed RNA secondary structure prediction. Methods. 52, 150-158 (2010).
  13. Das, R., Laederach, A., Pearlman, S. M., Herschlag, D., Altman, R. B. S. A. F. A. Semi-automated footprinting analysis software for high-throughput quantification of nucleic acid footprinting experiments. Rna-a Publication of the Rna Society. 11, 344-354 (2005).
  14. Kertesz, M., et al. Genome-wide measurement of RNA secondary structure in yeast. Nature. 467, 103-107 (2010).
  15. Underwood, J. G., et al. FragSeq: transcriptome-wide RNA structure probing using high-throughput sequencing. Nat. Methods. 7, 995-1001 (2010).
  16. Mauger, D. M., Weeks, K. M. Toward global RNA structure analysis. Nat. Biotechnol. 28, 1178-1179 (2010).
  17. Lucks, J. B., et al. Multiplexed RNA structure characterization with selective 2'-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq). Proc. Natl. Acad. Sci. USA. 108, 11063-11068 (2011).
  18. Vasa, S. M., Guex, N., Wilkinson, K. A., Weeks, K. M., Giddings, M. C. ShapeFinder: a software system for high-throughput quantitative analysis of nucleic acid reactivity information resolved by capillary electrophoresis. RNA. 14, 1979-1990 (2008).
  19. Reuter, J. S., Mathews, D. H. RNAstructure: software for RNA secondary structure prediction and analysis. BMC Bioinformatics. 11, 129 (2010).
  20. Pang, P. S., Elazar, M., Pham, E. A., Glenn, J. S. Simplified RNA secondary structure mapping by automation of SHAPE data analysis. Nucleic Acids Res. 39, e151 (2011).
  21. Deigan, K. E., Li, T. W., Mathews, D. H., Weeks, K. M. Accurate SHAPE-directed RNA structure determination. Proc. Natl. Acad. Sci. USA. 106, 97-102 (2009).
  22. Darty, K., Denise, A., Ponty, Y. VARNA: Interactive drawing and editing of the RNA secondary structure. Bioinformatics. 25, 1974-1975 (2009).
  23. Byun, Y., Han, K. PseudoViewer: web application and web service for visualizing RNA pseudoknots and secondary structures. Nucleic Acids Res. 34, 416-422 (2006).
  24. Brown, T., Brown, D. J. S., Eckstein, F. . Oligonucleotides and Analogues - A Practical Approach. , 20 (1990).
  25. Legiewicz, M., et al. The RNA Transport Element of the Murine musD Retrotransposon Requires Long-range Intramolecular Interactions for Function. J. Biol. Chem. 285, 42097-42104 (2010).
  26. Steen, K., Siegfried, N. A., Weeks, K. M. Syntheis of 1-methyl-8-nitroisatoic anhydride (1M7). Protocol Exchange. , (2011).
  27. Mortimer, S. A., Weeks, K. M. A fast-acting reagent for accurate analysis of RNA secondary and tertiary structure by SHAPE chemistry. J. Am. Chem. Soc. 129, 4144-4145 (2007).
  28. Mitra, S., Shcherbakova, I. V., Altman, R. B., Brenowitz, M., Laederach, A. High-throughput single-nucleotide structural mapping by capillary automated footprinting analysis. Nucleic Acids Res. 36, e63 (2008).
  29. Giddings, M. C., Severin, J., Westphall, M., Wu, J., Smith, L. M. A software system for data analysis in automated DNA sequencing. Genome Res. 8, 644-665 (1998).
  30. Aviran, S., et al. Modeling and automation of sequencing-based characterization of RNA structure. Proc. Natl. Acad. Sci. USA. 108, 11069-11074 (2011).

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