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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   

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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), RNA structure(RNA的结构), RNA chemical modification(RNA的化学改性), SHAPE(形状)

Material 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

  1. Cloning of RNA to be analyzed in SHAPE cassette
    RNA segment was cloned into SHAPE cassette (Figure 1) (Wang et al., 2010). SHAPE cassette has been checked to ensure that it is not prone to forming stable base pairing interactions with the internal sequence.
    We have design a structure cassette that contains flanking sequences that allow evaluate all positions within the RNA of interest. The primer binding site of this cassette efficiently binds to a cDNA primer.


    Figure 1. Schematic representation of the construct used for the determination of secondary RNA structure by SHAPE technology. The sequence fragment of the RNA to be analyzed is cloned between the EcoRI and HpaI restriction sites of the SHAPE cassette (Wang et al., 2010).

  2. Preparation of RNA by in vitro transcription
    1. Linearized DNA plasmid (≈0.5 µg) with SmaI used as template for in vitro transcription with MEGAshortscript (transcription protocol in user guide).
    2. After transcription, purification was made by phenol:chloroform extraction and alcohol precipitation as described in protocol. Alcohol precipitation was carried out overnight at - 20 °C with 3 M sodium acetate (pH 5.2) instead ammonium acetate.
    3. RNA recovery by centrifugation, followed by wash with 70% ethanol and dissolve pellet in 20 µl nuclease-free water.

  3. SHAPE analysis
    1. RNA refolding
      Need 500 ng to 1 µg RNA per reaction.
    2. Resuspend RNA in water. For example for 4 reactions resuspend RNA to make 40 µl total volume in an Eppendorf tube.
    3. Denaturation of RNA, 94 °C/1 min.
    4. Incubate on ice, 2 min.
    5. Divide 10 µl of refolded RNA into each tube.
    6. Add 30 µl of nuclease free water supplemented with 0.5 µl RNAse inhibitor into each tube.
    7. Add 10 µl of 5x SHAPE buffer and incubate, 30 °C/30 min.
    8. Modification
      Add 5.5 µl of DMSO (untreated) or BzCN (treated), 2 min.
    9. Add 6 µl of 3 M NaOAc (pH 5.2) in each epp.
    10. Add 2 µl of tRNA yeast at 1.9 mg/ml + 190 µl of 100% EtOH, -20 °C, overnight.
      Next day
    11. Centrifuge 14,000 rpm 4 °C, 40 min.
    12. Wash using 1ml of 70% ethanol and centrifuge 14,000 rpm, 4 °C, 10 min.
    13. Dry RNA pellet and resuspend in 20 µl. Quantify in Nanodrop. Continue with primer extension only with RNAs ≥ 100 ng/µl, 15 min.
    14. Label primer
      dH2O
      33 µl
      Primer (150 pmol)
      3 µl
      10x PNK buffer
      6 µl
      γP32 ATP
      15 µl
      PNK
      3 µl
      37 °C, 30 min
    15. Purify using biospin columns.
    16. Measure counts in a liquid scintillation counter if 107 cpm activity proceed to probing.
    17. Primer extension
      RNA modified

      Sequencing ladder

      RNA
      500 ng
      RNA no treated
      200 ng
      dNTPs (2.5 mM)
      2 µl
      dNTPs/ddNTPs
      2 µl
      Labeled primer
      2 µl
      Labeled primer
      2 µl
      dH2O



      Total
      12 µl
      Total
      12 µl
    18. Incubate on ice, 2 min.
    19. Incubate at room temperature, 5 min.
    20. Cocktail RT (reverse transcription)
      0.1 mM DTT
      2 µl
      RT buffer
      4 µl
      RNase inhibitor
      0.2 µl
      RT
      0.1 µl
      dH2O
      2 µl
      42 °C, 30 min
    21. Add 4 µl of 1 M NaOH, 95 °C, 30 min.
    22. Add of Gel loading buffer II (see Item 18 in Materials and Reagents) (15-20 µl), 95 °C, 4 min.
    23. Mix, spin and incubate on ice before loading onto the gel, 3 min.
    24. Pre-run gel: Load 2 µl Gel loading buffer II and conduct electrophoresis at constant power of 60 W, 15 min.
    25. Load samples in gel: 2 µl/lane. Conduct electrophoresis at constant power of 60 W.
    26. Run gel (you may do a long run approximately 3 h for resolving 5´end or a short run approx. 2 h for resolving 3’ end or your molecule), 2-3 h.
    27. Remove one of the glasses plates, allowing the gel to remain attached to the second plate. Press a sheet of Whatman paper on top of the gel. The gel will adhere to the Whatman paper and can be peeled away from the remaining glass plate. Cover the gel with plastic wrap.
    28. Dry the gel, 2 h.
    29. Expose to phosphorimager screen, ≈ 15 h.
    30. Scan in Typhoon 9410 scanner at 50 micron resolution (Figure 2), 10 min.
    31. Use SAFA Footprinting Software for analyze bands: https://simtk.org/docman/view.php/69/496/SAFAUserGuide_v1_1.pdf.
      The protocols implemented in SAFA have five steps: (a) lane identification, (b) gel rectification, (c) band assignment, (d) model fitting and (e) band-intensity normalization.
      Software will generate normalized bands intensities values (reactivity data).
    32. Use reactivity data from SAFA and classify it in “High, medium and low reactivity” according with normalized band intensity value from each nucleotide. Use “x” or “X” to indicate reactive nucleotides and “.” to indicate unreactive. Fill it in point 4 (Single-Stranded Chemical/Enzymatic Reactivity Data) of MC-Fold http://www.major.iric.ca/MC-Fold/ (Figure 3).
    33. MC-Fold will generate the top 20 structures.
    34. Choose structures that they are more fit of your reactivity data.


      Figure 2. Secondary structure probing of a cap-independent translation enhancer (3´-CITE). Structure probing by SHAPE of the first 65 nt of the 3´-UTR of MNSV-N, including the new 3´-CITE. Primer extension products separated on denaturing PAGE of RNA treated (lanes 4-6) or untreated (lane 3) with BzCN. Concentrations of Mg2+ (mM) are indicated above lines 4-6. The sequencing ladder was generated by reverse transcription of unmodified RNA in the presence of dideoxyCTP (ddCTP) or ddATP. Positions of nucleotides A4002, A4029 and A4047 are indicated on the left.


      Figure 3. Secondary structure of the new 3´-CITE probed in Figure 2. SHAPE reactivity of nucleotides superimposed on secondary structure predicted by Mfold. Color-coded bases indicate the levels of BzCN modification, with warmer color indicating greater modification (inset).

Recipes

  1. 10 x TBE buffer
    108 g of Tris base
    55 g of boric acid
    40 ml of 0.5 M of EDTA (pH 8.0)
  2. Denaturing polyacrylamide gel electrophoresis (PAGE)
    Gel sequencing system
    8 M urea
    8-15% 37% acrylamide-bisacrylamide 29:1
    1x TBE
    10% ammonium persulfate (APS)
    N, N, N´, N´-tetramethylethylenediamine (TEMED)
    Note: Alternatively, The UreaGel System can be used to prepare gels of varying percentage.
  3. Sequencing ladders (ddNTP/dNTP mix)
    Note: If signal is too strong add less ddNTP and vice versa.

    Sequencing ladders

    A (ul)
    T (ul)
    C (ul)
    G (ul)
    dATP (10 mM)
    1
    2
    2
    2
    dTTP
    2
    0.5
    2
    2
    dCTP
    2
    2
    1
    2
    dGTP
    2
    2
    2
    1
    ddATP (5 mM)
    20



    ddTTP

    20


    ddCTP


    10

    ddGTP



    20
    dH2O
    13
    13.5
    23
    13

  4. 5x SHAPE buffer
    Note: The buffer should be optimized for each individual RNA to be studied.


    1x
    5x
    KCl
    100 mM
    20 ml 1 M KCl
    HEPES KOH (pH 7.5)
    50 mM
    10 ml 1 M HEPES KOH (pH 7.5)
    MgCl2
    8 mM
    1.6 ml 1 M MgCl2


    8.4 ml H2O

  5. 0.4 M benzoyl cyanide
    Weight 0.0524 g and dissolve in 1 ml of DMSO

Acknowledgments

This work was supported by grants AGL2009-07552/AGR from Ministerio de Ciencia e Innovación (Spain) and EUI2009-04009 of the transnational (Germany, France, Spain and Portugal) cooperation within the 2009 PLANT-KBBE initiative with funding from Ministerio de Ciencia e Innovación (Spain). Manuel Miras was recipient of a predoctoral fellowship from Ministerio de Ciencia e Innovación (Spain). JJK was funded by grant 2011-67012-30715 from the USDA National Research Initiative.
This protocol was adapted from previous work (Wilkinson et al., 2006; Mortimer and Weeks, 2007).

References

  1. Kraft, J. J., Treder, K., Peterson, M. S. and Miller, W. A. (2013). Cation-dependent folding of 3' cap-independent translation elements facilitates interaction of a 17-nucleotide conserved sequence with eIF4G. Nucleic Acids Res 41(5): 3398-3413.
  2. Miras, M., Sempere, R. N., Kraft, J. J., Miller, W. A., Aranda, M. A. and Truniger, V. (2014). Interfamilial recombination between viruses led to acquisition of a novel translation‐enhancing RNA element that allows resistance breaking. New Phytol 202(1): 233-246.
  3. Mortimer, S. A. and Weeks, K. M. (2007). A fast-acting reagent for accurate analysis of RNA secondary and tertiary structure by SHAPE chemistry. J Am Chem Soc 129(14): 4144-4145.
  4. Wang, Z., Kraft, J. J., Hui, A. Y. and Miller, W. A. (2010). Structural plasticity of Barley yellow dwarf virus-like cap-independent translation elements in four genera of plant viral RNAs. Virology 402(1): 177-186.
  5. Wang, Z., Parisien, M., Scheets, K. and Miller, W. A. (2011). The cap-binding translation initiation factor, eIF4E, binds a pseudoknot in a viral cap-independent translation element. Structure 19(6): 868-880.
  6. Wilkinson, K. A., Merino, E. J. and Weeks, K. M. (2006). Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE): quantitative RNA structure analysis at single nucleotide resolution. Nat Protoc 1(3): 1610-1616.

简介

该方案描述了使用通过引物延伸(缩写为SHAPE)分析的选择性2'-羟基酰化来确定溶液中RNA片段的二级结构的方法。 其包括使用苯甲酰氰(BzCN)以序列非依赖性方式非常快速地化学修饰柔性且因此可能的单链核苷酸,形成2'-O-加合物。 然后通过引物延伸分析RNA中的修饰。 逆转录酶被形成的2'-O-加合物阻断。 该方法的优点是,首先,不是每个RNA分子研究,而是在延伸反应中使用的引物被标记,其次,在测序凝胶中分析得到的cDNA比修饰的RNA更稳定。

关键字:RNA二级结构, RNA的结构, RNA的化学改性, 形状

材料和试剂

  1. 酵母tRNA(Life Technologies,Ambion ,目录号:AM7119)
  2. MEGAshortscript T7转录试剂盒(Life Technologies,Ambion ,目录号:AM1354)
  3. 寡核苷酸(50μM)
  4. 重组RNA酶抑制剂(Takara Bio Company,目录号:2313A)
  5. 氯化钾(Sigma-Aldrich,目录号:P9541)
  6. HEPES(Sigma-Aldrich,目录号:H4034)
  7. 氯化镁(Sigma-Aldrich,目录号:63063)
  8. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D8418)
  9. 苯甲酰氰(Sigma-Aldrich,目录号:115959) 注意:保存在干燥器中!
  10. 3 M乙酸钠(pH 5.5)(Life Technologies,Ambion ,目录号:AM9740)
  11. T4多核苷酸激酶(New England Biolabs,目录号:M0201S)
  12. ATP([γ-32 P] -6,000Ci/mmol 10mCi/ml铅,250μCi)(PerkinElmer,目录号:NEG002Z250UC)
  13. Micro Bio-Spin P-30凝胶柱Tris缓冲液(Bio-Rad Laboratories,目录号:732-6250)
  14. 用于引物延伸的dNTP混合物(各2.5mM)(Takara Bio公司,目录号:4030)
  15. ddNTP(设定为5mM)(GE Healthcare,目录号:27-2045-01)
  16. dNTP(梯度设置为100mM稀释至10mM)(Life Technologies,目录号10297-018)
  17. SuperScript II(Life Technologies,Invitrogen TM,目录号:18064-014)
  18. 凝胶加载缓冲液II(Life Technologies,Ambion ,目录号:AM8546G)
  19. 氢氧化钠(Sigma-Aldrich,目录号:221465)
  20. 过硫酸铵(APS)(Bio-Rad Laboratories,目录号:161-0700)
  21. 100%乙醇
  22. Biospin柱(Bio-Rad Laboratories,目录号:732-6250)
  23. 10x TBE缓冲区(参见配方)
  24. 变性聚丙烯酰胺凝胶电泳(PAGE)(参见配方)
  25. 排序梯(ddNTP/dNTP混合)(参见配方)
  26. 5x SHAPE缓冲区(请参阅配方)
  27. 0.4 M苯甲酰氰(见配方)

设备

  1. 标准实验室设备
  2. 纳米装置
  3. 培养箱或水浴
  4. 测序凝胶电泳系统
  5. 电源
  6. 真空泵
  7. 凝胶干燥器(Bio-Rad Laboratories,型号:583)
  8. 磷化仪和屏幕
  9. 台风9410扫描仪
  10. UreaGel系统(国家诊断,目录号:EC-833)

软件

  1. 图像分析软件(Bio-Rad Laboratories,Quantity One; SAFA footprinting software, https://simtk.org/home/safa
  2. RNA二级结构预测
    1. MC-Fold: http://www.major.iric.ca/MC-Fold
    2. Mfold: http://mfold.rna.albany.edu/?q = mfold/RNA-折叠形式

程序

  1. 克隆要在SHAPE盒中分析的RNA
    RNA片段克隆到SHAPE盒中(图1)(Wang等人,2010)。已经检查了SHAPE盒以确保其不容易与内部序列形成稳定的碱基配对相互作用。
    我们设计了包含侧翼序列的结构盒,其允许评估感兴趣的RNA内的所有位置。该盒的引物结合位点有效地结合到cDNA引物

    图1.用于通过SHAPE技术测定二级RNA结构的构建体的示意图。将要分析的RNA的序列片段克隆在EcoRI和HpaI之间 SHAPE盒的限制性位点(Wang等人,2010)。

  2. 通过体外转录制备RNA
    1. 线性化DNA质粒(≈0.5μg),用SmaI作为模板用于体外转录使用MEGAshortscript(转录方案在用户 指南)。
    2. 转录后,通过纯化 苯酚:氯仿萃取和醇沉淀 协议。 在-20℃下进行乙醇沉淀过夜 用3M乙酸钠(pH 5.2)代替乙酸铵。
    3. 通过离心回收RNA,然后用70%乙醇洗涤,并将沉淀溶于20μl无核酸酶的水中

  3. SHAPE分析
    1. RNA重折叠
      每次反应需要500ng至1μgRNA。
    2. 在水中重悬RNA。 例如对于4个反应,重悬RNA以在Eppendorf管中制备40μl总体积
    3. RNA变性,94℃/1分钟
    4. 在冰上孵育2分钟。
    5. 将10μl重折叠的RNA分到每个管中
    6. 向每个管中加入30μl不含核酸酶的水,其中补充有0.5μlRNA酶抑制剂
    7. 加入10μl5x SHAPE缓冲液,并孵育,30℃/30分钟
    8. 修改
      加入5.5μlDMSO(未处理)或BzCN(处理),2分钟
    9. 在每个epp中加入6μl的3M NaOAc(pH5.2)
    10. 加入2μl的tRNA酵母1.9毫克/毫升+ 190微升100%乙醇,-20°C,过夜 第二天
    11. 14,000rpm离心4℃,40分钟
    12. 使用1ml的70%乙醇洗涤,并在14,000rpm,4℃,10分钟离心
    13. 干燥RNA沉淀并重悬于20μl。 在Nanodrop中量化。 继续引物延伸只有RNAs≥100 ng /μl,15分钟。
    14. 标签引物
      dH 2 2 O 33微升
      引物(150pmol)
      3微升
      10x PNK缓冲区
      6微升
      γP32ATP
      15微升
      PNK
      3微升
      37℃,30分钟
    15. 使用biospin柱进行净化。
    16. 如果10 cpm活性进行探测,在液体闪烁计数器中测量计数。
    17. 引物延伸
      RNA修饰

      顺序梯子

      RNA
      500 ng
      RNA未经处理
      200 ng
      dNTPs(2.5mM) 2微升
      dNTPs/ddNTPs
      2微升
      标签引物
      2微升
      标签引物
      2微升
      dH 2 2 O


      总计
      12微升
      总计
      12微升
    18. 在冰上孵育2分钟。
    19. 在室温下孵育5分钟
    20. 鸡尾酒RT(逆转录)
      0.1 mM DTT
      2微升
      RT缓冲区
      4微升
      核糖核酸酶抑制剂
      0.2μl
      RT
      0.1μl
      dH 2 2 O 2微升
      42℃,30分钟
    21. 加入4μl1M NaOH,95℃,30分钟
    22. 加入凝胶上样缓冲液II(参见材料和试剂中的第18项)(15-20μl),95℃,4分钟。
    23. 混合,旋转并在加载到凝胶上之前在冰上孵育3分钟
    24. 预运行凝胶:加载2μl凝胶上样缓冲液II,并以60W,15分钟的恒定功率进行电泳
    25. 加载凝胶样品:2μl/泳道。 以60 W的恒定功率进行电泳
    26. 运行凝胶(你可以长时间运行约3小时解决 5秒或短跑约。 2小时用于拆分3'末端或分子), 2-3小时。
    27. 取出一个眼镜板,让凝胶 保持附接到第二板。 按一张Whatman纸 凝胶顶部。 凝胶将粘附在Whatman纸上并且可以 从剩余的玻璃板剥离。 用塑料覆盖凝胶 包装。
    28. 干燥凝胶,2小时
    29. 暴露于phosphorimager屏幕,≈15小时。
    30. 在Typhoon 9410扫描仪中以50微米分辨率(图2),10分钟扫描
    31. 使用SAFA Footprinting软件进行分析频段: https://simtk.org/docman /view.php/69/496/SAFAUserGuide_v1_1.pdf。
      在SAFA中实现的协议具有五个步骤:(a)通道 鉴定,(b)凝胶精馏,(c)谱带分配,(d)模型 拟合和(e)频带强度归一化。
      软件将生成标准化带强度值(反应性数据)。
    32. 使用SAFA的反应性数据,并将其分类为"高,中和 低反应性"根据来自每个的标准化带强度值  核苷酸。使用"x"或"X"表示反应性核苷酸,使用"。"表示 表示无反应。填写点4(单股 化学/酶反应性数据) http://www.major.iric.ca/MC-Fold/(图3)。
    33. MC-Fold将生成前20个结构。
    34. 选择他们更适合您的反应数据的结构。


      图2.帽独立翻译的二级结构探测 增强子(3'-CITE)。 SHAPE的前65个结构探针  3'-UTR,包括新的3'-CITE。引物延伸产物 在RNA处理的(泳道4-6)或未处理的变性PAGE上分离 (泳道3)。 Mg 2+的浓度(mM)如上所示 第4-6行。通过逆转录产生测序梯 的未修饰的RNA在双脱氧CTP(ddCTP)或ddATP的存在下。 核苷酸A4002,A4029和A4047的位置显示在 剩下。


      图3.新3'-CITE探针的二级结构 图2。叠加在二级核苷酸上的核苷酸的SHAPE反应性 结构由Mfold预测。颜色编码的碱基表示的水平 BzCN改性,具有更暖的颜色,表明更大的改性 (插图)。

食谱

  1. 10 x TBE缓冲区
    108克Tris碱
    55克硼酸 40ml 0.5M EDTA(pH8.0)
  2. 变性聚丙烯酰胺凝胶电泳(PAGE)
    凝胶测序系统
    8 M尿素
    8-15%37%丙烯酰胺 - 双丙烯酰胺29:1
    1x TBE
    10%过硫酸铵(APS)
    N,N,N',N'-四甲基乙二胺(TEMED) 注意:或者,UreaGel系统可用于制备不同百分比的凝胶。
  3. 顺序梯子(ddNTP/dNTP mix)
    注意:如果信号太强,则添加较少的ddNTP,反之亦然。

    排序梯子

    A(ul)
    T(ul)
    C(ul)
    G(ul)
    dATP(10mM) 1
    2
    2
    2
    dTTP
    2
    0.5
    2
    2
    dCTP
    2
    2
    1
    2
    dGTP
    2
    2
    2
    1
    ddATP(5mM) 20



    ddTTP

    20


    ddCTP


    10

    ddGTP



    20
    dH 2 2 O 13
    13.5
    23
    13

  4. 5x SHAPE缓冲区
    注意:应针对每个要研究的单个RNA优化缓冲液。


    1x
    5x
    KCl
    100 mM
    20ml 1 M KCl
    HEPES KOH(pH 7.5)
    50 mM
    10ml 1M HEPES KOH(pH 7.5)
    MgCl 2
    8 mM
    1.6ml 1M MgCl 2·h/v


    8.4ml H 2 O x/v

  5. 0.4M苯甲酰氰 重量0.0524g并溶于1ml DMSO中

致谢

这项工作得到了2009年PLANT-KBBE行动计划下的跨国(德国,法国,西班牙和葡萄牙)合作的授权AGL2009-07552/AGR和西班牙Ministerion deInnovacón(西班牙)的EUI2009-04009资助, eInnovación(西班牙)。 Manuel Miras是西班牙Ministerio de Ciencia eInnovación的牧师奖学金获得者。 JJK由美国农业部国家研究计划的资助2011-67012-30715资助 该方案改编自以前的工作(Wilkinson等人,2006; Mortimer和Weeks,2007)。

参考文献

  1. Kraft,J.J.,Treder,K.,Peterson,M.S.and Miller,W.A。(2013)。 3'帽独立翻译元件的阳离子依赖性折叠促进17核苷酸保守序列的相互作用with eIF4G。 Nucleic Acids Res 41(5):3398-3413。
  2. Miras,M.,Sempere,R.N.,Kraft,J.J.,Miller,W.A.,Aranda,M.A.and Truniger,V。(2014)。 病毒间的家族间重组导致获得了一种新的翻译增强RNA元件,允许抗性破坏。 New Phytol 202(1):233-246。
  3. Mortimer,S.A。和Weeks,K.M。(2007)。 用于通过SHAPE化学准确分析RNA二级和三级结构的快速作用试剂。 J Am Chem Soc 129(14):4144-4145。
  4. Wang,Z.,Kraft,J.J.,Hui,A.Y。和Miller,W.A。(2010)。 四种植物病毒RNA中大麦黄矮星病毒样帽子非依赖性翻译元件的结构可塑性 。 Virology 402(1):177-186。
  5. Wang,Z.,Parisien,M.,Scheets,K.and Miller,W.A。(2011)。 帽结合性翻译起始因子eIF4E结合病毒帽独立翻译元件中的假性结节 。 结构 19(6):868-880。
  6. Wilkinson,K.A.,Merino,E.J.and Weeks,K.M。(2006)。 通过引物延伸(SHAPE)分析的选择性2'-羟基酰化:单核苷酸的定量RNA结构分析 Nat Protoc 1(3):1610-1616。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
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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