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Determination of the Secondary Structure of an RNA fragment in Solution: Selective 2`-Hydroxyl Acylation Analyzed by Primer Extension Assay (SHAPE)

溶液中RNA片段二级机构的测定：引物延伸法（SHAPE）分析选择性2-羟基酰化

MM Manuel Miras
RS Raquel N. Sempere
JK Jelena J. Kraft
WM W. Allen Miller
MA Miguel A. Aranda
VT Veronica Truniger

发布: 2015年01月20日第5卷第2期 DOI: 10.21769/BioProtoc.1386 浏览次数: 13699

评审: Tie LiuAnonymous reviewer(s)

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Abstract

This protocol describes the methodology for the determination of the secondary structure of an RNA fragment in solution using Selective 2´-Hydroxyl Acylation analyzed by Primer Extension, abbreviation SHAPE. It consists in the very fast chemical modification of flexible and therefore possibly single-stranded nucleotides in a sequence-independent manner using benzoyl cyanide (BzCN), forming 2´-O-adducts. The modifications in the RNA are then analyzed by primer extension. Reverse transcriptase is blocked by the 2´-O-adducts formed. The advantage of the method is, first, that not each RNA molecule studied but the primer used in the extension reaction is labelled and, second, that the resulting cDNA analyzed in sequencing gels is much more stable than the modified RNA.

Keywords: RNA secondary structure (RNA二级结构) search RNA structure (RNA的结构) search RNA chemical modification (RNA的化学改性) search SHAPE (形状) search

Materials and Reagents

  1. Yeast tRNA (Life Technologies, Ambion®, catalog number: AM7119 )
  2. MEGAshortscript T7 Transcription Kit (Life Technologies, Ambion®, catalog number: AM1354 )
  3. Oligonucleotide (50 μM)
  4. Recombinant RNase inhibitor (Takara Bio Company, catalog number: 2313A )
  5. Potassium chloride (Sigma-Aldrich, catalog number: P9541 )
  6. HEPES (Sigma-Aldrich, catalog number: H4034 )
  7. Magnesium chloride (Sigma-Aldrich, catalog number: 63063 )
  8. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8418 )
  9. Benzoyl cyanide (Sigma-Aldrich, catalog number: 115959 )
    Note: Keep in desiccator!
  10. 3 M sodium acetate (pH 5.5) (Life Technologies, Ambion®, catalog number: AM9740 )
  11. T4 Polynucleotide kinase (New England Biolabs, catalog number: M0201S )
  12. ATP ([γ-32P]- 6,000 Ci/mmol 10 mCi/ml Lead, 250 µCi) (PerkinElmer, catalog number: NEG002Z250UC )
  13. Micro Bio-Spin P-30 Gel Columns Tris Buffer (Bio-Rad Laboratories, catalog number: 732-6250 )
  14. dNTPs mixture for primer extension (2.5 mM each) (Takara Bio Company, catalog number: 4030 )
  15. ddNTPs (set 5 mM) (GE Healthcare, catalog number: 27-2045-01 )
  16. dNTPs (set 100 mM diluted to 10 mM for ladder) (Life Technologies, catalog number 10297-018 )
  17. SuperScript II (Life Technologies, InvitrogenTM, catalog number: 18064-014 )
  18. Gel loading buffer II (Life Technologies, Ambion®, catalog number: AM8546G )
  19. Sodium hydroxide (Sigma-Aldrich, catalog number: 221465 )
  20. Ammonium persulfate (APS) (Bio-Rad Laboratories, catalog number: 161-0700 )
  21. 100% ethanol
  22. Biospin columns (Bio-Rad Laboratories, catalog number: 732-6250)
  23. 10x TBE buffer (see Recipes)
  24. Denaturing polyacrylamide gel electrophoresis (PAGE) (see Recipes)
  25. Sequencing ladders (ddNTP/dNTP mix) (see Recipes)
  26. 5x SHAPE buffer (see Recipes)
  27. 0.4 M benzoyl cyanide (see Recipes)

Equipment

  1. Standard laboratory equipment
  2. Nanodrop device
  3. Incubator or water bath
  4. Sequencing gel electrophoresis system
  5. Power supply
  6. Vacuum pump
  7. Gel dryer (Bio-Rad Laboratories, model: 583 )
  8. Phosphorimaging instrument and screen
  9. Typhoon 9410 scanner
  10. The UreaGel System (National Diagnostics, catalog number: EC-833 )

Software

  1. Image analysis software (Bio-Rad Laboratories, Quantity One; SAFA footprinting software, https://simtk.org/home/safa)
  2. RNA secondary structure prediction
    1. MC-Fold: http://www.major.iric.ca/MC-Fold
    2. Mfold: http://mfold.rna.albany.edu/?q=mfold/RNA-Folding-Form

Procedure

English
中文翻译

文章信息

版权信息

© 2015 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Miras, M., Sempere, R. N., Kraft, J. J., Miller, W. A., Aranda, M. A. and Truniger, V. (2015). Determination of the Secondary Structure of an RNA fragment in Solution: Selective 2`-Hydroxyl Acylation Analyzed by Primer Extension Assay (SHAPE). Bio-protocol 5(2): e1386. DOI: 10.21769/BioProtoc.1386.

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分类

微生物学 > 微生物-宿主相互作用 > 病毒

植物科学 > 植物分子生物学 > RNA

分子生物学 > RNA > RNA 结构

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