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The ribonuclease H (RNase H) polymerase-independent cleavage assay allows detection and quantification of RNase H activity of reverse transcriptase (RT) enzymes with a hybrid substrate formed by a fluorescein labeled RNA annealed with Dabcyl DNA (Figure 1). Here we describe a protocol that we have adapted for HIV-1 RT expressed from a p(His)6-tagged p66/p51 HIV-1HXB2 RT-prot plasmid and for RT of the prototype foamy virus (PFV RT).



Figure 1. Scheme of the principle of the experiment. The RNA substrate (blue) labeled with the fluorophore fluorescein (F, yellow) is annealed with complementary DNA strand (green) labeled with a quencher molecule Dabcyl (D, red). Panel A. In the intact substrate the quencher is so close to the fluorophore that it can quench the fluorescence emitted after excitation. Panel B. After the RNA substrate is cut by the RNase H a few ribonucleotides oligo labeled with the fluorescein is free to escape from the quencher, and to release fluorescence after excitation.

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RNase H Polymerase-independent Cleavage Assay for Evaluation of RNase H Activity of Reverse Transcriptase Enzymes
RNase H 聚合酶依赖性分裂试验评估逆转录酶中Rnase H的活性

微生物学 > 抗微生物试验 > 抗病毒试验
作者: Angela Corona
Angela CoronaAffiliation: Department of Life and Environmental Sciences, University of Cagliari, Cittadella Universitaria di Monserrato, Monserrato , Italy
Bio-protocol author page: a2469
 and Enzo Tramontano
Enzo TramontanoAffiliation: Department of Life and Environmental Sciences, University of Cagliari, Cittadella Universitaria di Monserrato, Monserrato, Italy
For correspondence: tramon@unica.it
Bio-protocol author page: a2470
Vol 5, Iss 16, 8/20/2015, 2153 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1561

[Abstract] The ribonuclease H (RNase H) polymerase-independent cleavage assay allows detection and quantification of RNase H activity of reverse transcriptase (RT) enzymes with a hybrid substrate formed by a fluorescein labeled RNA annealed with Dabcyl DNA (Figure 1). Here we describe a protocol that we have adapted for HIV-1 RT expressed from a p(His)6-tagged p66/p51 HIV-1HXB2 RT-prot plasmid and for RT of the prototype foamy virus (PFV RT).



Figure 1. Scheme of the principle of the experiment. The RNA substrate (blue) labeled with the fluorophore fluorescein (F, yellow) is annealed with complementary DNA strand (green) labeled with a quencher molecule Dabcyl (D, red). Panel A. In the intact substrate the quencher is so close to the fluorophore that it can quench the fluorescence emitted after excitation. Panel B. After the RNA substrate is cut by the RNase H a few ribonucleotides oligo labeled with the fluorescein is free to escape from the quencher, and to release fluorescence after excitation.
Keywords: RNase H assay(RNase H测定), Drug Screening(药物筛选), Ribonuclease activity(核糖核酸酶活性), RNase H inhibitors (RNase H抑制剂), HIV-1 RT RNase H(HIV-1 RT RNA酶H)

[Abstract] 核糖核酸酶H(RNase H)聚合酶非依赖性切割测定允许用由与Dabcyl DNA退火的荧光素标记的RNA形成的杂交底物检测和定量逆转录酶(RT)酶的RNA酶H活性(图1)。在这里我们描述了一个协议,我们已适应的艾滋病毒1 RT表示从p(His)6标记的p66/p51艾滋病毒1 HXB2 RT-prot质粒和原型泡沫病毒(PFV RT)RT。



Materials and Reagents

  1. Reverse transcriptase enzyme (HIV-1 RT or PFV RT). HIV-1 RT expressed and purified from p(His)6-tagged p66/p51 HIV-1HXB2 RT-prot plasmid (Corona et al., 2014a), PFV RT provided by Birgitta M. Wöhrl from Universität Bayreuth, Lehrstuhl Biopolymere, Bayreuth, Germany (Corona et al., 2014b).
  2. Distilled water (DNase/RNase-free UltraPureTM) (Life Technologies, InvitrogenTM, catalog number: 10977-015 )
  3. 5'-GAUCUGAGCCUGGGAGCU-FLUORESCEIN-3' (HPLC, dry, QC: Mass Check) (Metabion)
  4. 5'-DABCYL-AGCTCCCAGGCTCAGATC-3' (HPLC, dry, QC: Mass Check) (Metabion)
  5. 1 M KCl solution (Sigma-Aldrich, catalog number: 60142-100ML-F )
  6. 1 M MgCl2 solution (Sigma-Aldrich, catalog number: 63069-100ML )
  7. 1 M Dithiothreitol (DTT) solution (Life Technologies, InvitrogenTM, catalog number: P2375 )
  8. 1 M Tris-HCl Solution (pH 7.8) prepared from Trizma base® (Sigma-Aldrich, catalog number 93352 )
  9. 1 M Tris-HCl Solution (pH 8.1) prepared from Trizma base® (Sigma-Aldrich, catalog number: 93352)
  10. 5 M NaCl solution (Life Technologies, InvitrogenTM, catalog number: AM9760G )
  11. EDTA (0.5 M, pH 8.0) UltraPureTM (Life Technologies, InvitrogenTM, catalog number: 15575-020 )
  12. Dimethyl sulfoxide (DMSO) >=99.5% (Sigma-Aldrich, catalog number: D5879-1 L )
  13. Microcentrifuge tubes, extended capacity, volume 0.65 ml, graduated, siliconized polypropylene (Sigma-Aldrich, catalog number: T3406-250EA )
  14. OPTIPLATE-96 F /50B (PerkinElmer, catalog number: 6005270 )
  15. HIV-1 RT reaction mix (1.25x) (see Recipes)
  16. PFV RT reaction mix (1.25x) (see Recipes)

Equipment

  1. Thermal cycler
  2. Refrigerated tabletop microcentrifuge
  3. Sonicator Bath (Elma Ultrasonic bath Transonic T 310)
  4. Vortex mixer
  5. Multi Thermo-Shaker for microplates (Bionics)
  6. Multilabel counter plate reader Victor 3 (PerkinElmer, model: 1420-051 ) equipped with filters for fluorescein fluorophore 490/528 nm (excitation/emission wavelength)

Procedure

  1. Preparation of the RNA/DNA hybrid as the substrate
    1. Resuspend carefully each RNA and DNA oligos at final concentration of 500 µM in 100 mM Tris-HCl (pH 8.1), 0.5 mM EDTA (pH 8.0).
    2. Prepare an annealing solution of 50 µM final of both RNA and DNA oligos with 50 mM NaCl in DNase/RNase-free water then aliquot 50 µl in a 0.2 ml PCR tube. Vortex carefully and then and then spin down briefly at 5,000 rpm to allow all the liquid to lay in the bottom of the tube.
    3. Do not overlay mineral oil on the solution. Place the tube in a thermal cycler and set up a program to perform the following profile:
      Heat to 95 °C and remain at 95 °C for 2 min;
      Ramp cool to 25 °C over a period of 45 min;
      Proceed to a storage temperature of 4 °C.
    4. The RNA/DNA hybrid solution (50 μM) is ready to be used (see Recipes). The solution can be stored at -20 °C.

  2. Preparation of dilutions of inhibitors
    1. Dissolve the powder of compound in DMSO at 10 mM final concentration, sonicate it (e.g.: 10” at 50 Hz) and warm it if needed (e.g.: 2’ 45 °C), to be sure all the compound goes in solution.
    2. Starting from the 10 mM solution in DMSO make the serial dilutions of compound in Milli-Q purified water: Starting from 1 mM dilution and making three fold dilution 10 fold more concentrate with respect to the final concentration needed. e.g.: 1 mM -> 333.33 µM->111.11 µM dilution will result 100 µM->33.33 µM -> 11.11 µM in the samples. Please see Notes.

  3. RNase H assay
    1. Plan an experiment without RT enzymes (blank), with the RT enzyme in the absence of inhibitors (control), and in the presence of increasing concentration of inhibitors (samples). It is better to use a wide range of concentrations to have a better curve of inhibition. The reaction volume is 100 µl.
    2. In one OPTIPLATE-96 F/50B, first add water (20 µl in blanks and 10 µl in controls).
    3. Add 80 µl of RT reaction mix (1.25x).
    4. Add 10 µl of the inhibitor dilution at the established concentration in the samples (see Figure 2 as an example).
    5. Finally add 10 µl of the enzyme (20 ng of HIV-1 RT) or (2 nM of PFV RT) to each well (control and samples).
    6.  Cover the plate with the lid and incubate the reaction for 1 h at 37 °C at 200 rpm in the Multi Thermo-Shaker for microplates.
    7. Stop the reaction by addition of 50 µl EDTA (0.5 M, pH 8.0) to each well.
    8. Quantify the reaction with the Victor 3 at 490/528 nm (excitation/emission wavelength).
    9.  Elaborate the results by subtracting the value of the blank from all the samples and reporting the obtained values in the samples with the inhibitor as a percentage of the control.
    10. Calculate by interpolation the IC50 value of the inhibitors as the concentration that reports a 50% of reduction of the signal of the product respect to the control.

Representative data



Figure 2. The results from three independent experiments (biological replicates) are shown as percentage of control. Error bars represent the standard deviation.

Notes

  1. This protocol has been applied to the HIV-1 and PFV RT enzymes. However, it can be used with virtually any RT enzymes with an RNase H activity.
  2. We recommend to make the inhibitor dilutions in water since the DMSO solvent affects the RNase H activity so its concentration in the sample should never overcome 1.01%.

Recipes

  1. Reaction mix (1.25x)
    For HIV-1 RT:
    62.5 mM Tris-HCl (pH 7.8)
    7.25 M MgCl2
    1.25 M DTT
    100 mM KCl
    0.3125 µM of RNA/DNA hybrid
  2. PFV RT reaction mix (1.25x)
    62.5 mM Tris-HCl (pH 8.1)
    7.25 mM MgCl2
    1.25 mM DTT
    100 mM KCl
    0.3125 µM of RNA/DNA hybrid

Acknowledgments

The p(His)6-tagged p66/p51 HIV-1HXB2 RT-prot plasmid was kindly provided by Stuart Le Grice Laboratory (NCI Frederick). The foamy virus reverse transcriptase was kindly provided by Birgitta M. Wöhrl Universität Bayreuth, Lehrstuhl Biopolymere, Bayreuth, Germany. This protocol was modified and adapted from Parniak et al. (2003).

References

  1. Corona, A., Di Leva, F. S., Thierry, S., Pescatori, L., Cuzzucoli Crucitti, G., Subra, F., Delelis, O., Esposito, F., Rigogliuso, G., Costi, R., Cosconati, S., Novellino, E., Di Santo, R. and Tramontano, E. (2014a). Identification of highly conserved residues involved in inhibition of HIV-1 RNase H function by Diketo acid derivatives. Antimicrob Agents Chemother 58(10): 6101-6110.
  2. Corona, A., Schneider, A., Schweimer, K., Rösch, P., Wöhrl, B. M. and Tramontano, E. (2014b). Inhibition of foamy virus reverse transcriptase by human immunodeficiency virus type 1 RNase H inhibitors. Antimicrob Agents Chemother 58(7): 4086-4093.
  3. Parniak, M. A., Min, K. L., Budihas, S. R., Le Grice, S. F. and Beutler, J. A. (2003). A fluorescence-based high-throughput screening assay for inhibitors of human immunodeficiency virus-1 reverse transcriptase-associated ribonuclease H activity. Anal Biochem 322(1): 33-39.

材料和试剂

  1. 逆转录酶(HIV-1 RT或PFV RT)。 从p(His)6标记的p66/p51 HIV-1HXB2 RT-prot质粒(Corona等人,2014a)表达和纯化的HIV-1 RT,由BirgittaM.Wöhrl提供的PFV RT UniversitätBayreuth,Lehrstuhl Biopolymere,Bayreuth,Germany(Corona等人,2014b)。
  2. 蒸馏水(DNase/RNase-free UltraPure TM)(Life Technologies,Invitrogen TM,目录号:10977-015)
  3. 5'-GAUCUGAGCCUGGGAGCU-FLUORESCEIN-3'(HPLC,干燥,QC:质量检查)(Metabion)
  4. 5'-DABCYL-AGCTCCCAGGCTCAGATC-3'(HPLC,干燥,QC:质量检查)(Metabion)
  5. 1M KCl溶液(Sigma-Aldrich,目录号:60142-100ML-F)
  6. 1M MgCl 2溶液(Sigma-Aldrich,目录号:63069-100ML)
  7. 将1M二硫苏糖醇(DTT)溶液(Life Technologies,Invitrogen TM,目录号:P2375)
  8. 由Trizma base (Sigma-Aldrich,目录号93352)制备的1M Tris-HCl溶液(pH7.8)
  9. 由Trizma base (Sigma-Aldrich,目录号:93352)制备的1M Tris-HCl溶液(pH 8.1)
  10. 5 M NaCl溶液(Life Technologies,Invitrogen ,目录号:AM9760G)
  11. EDTA(0.5M,pH 8.0)UltraPure TM(Life Technologies,Invitrogen TM,目录号:15575-020)
  12. 二甲基亚砜(DMSO)≥99.5%(Sigma-Aldrich,目录号:D5879-1L)
  13. 微量离心管,扩展容量,体积0.65ml,刻度,硅化聚丙烯(Sigma-Aldrich,目录号:T3406-250EA)
  14. OPTIPLATE-96F/50B(PerkinElmer,目录号:6005270)
  15. HIV-1 RT反应混合物(1.25x)(参见配方)
  16. PFV RT反应混合物(1.25x)(参见配方)

设备

  1. 热循环仪
  2. 制冷台式微量离心机
  3. 超声波浴(Elma超声波浴Transonic T 310)
  4. 涡流搅拌器
  5. 用于微孔板(Bionics)的多功能振动器
  6. 配有用于荧光荧光团490/528nm(激发/发射波长)的过滤器的多标记计数板读数器Victor 3(PerkinElmer,型号:1420-051)

程序

  1. 制备作为底物的RNA/DNA杂交体
    1. 重新悬浮每个RNA和DNA寡核苷酸的最终浓度   500μM,在100mM Tris-HCl(pH 8.1),0.5mM EDTA(pH 8.0)中。
    2. 制备50μM终浓度的RNA和DNA寡核苷酸的退火溶液 用50mM NaCl的DNase/RNase-free水,然后等分50μl在0.2 ml PCR管。 小心涡旋,然后在下旋转 5000转/分,以使所有的液体放置在管的底部
    3. 不要在溶液上覆盖矿物油。 将管放置在热   循环仪并设置程序以执行以下配置文件:
      加热至95℃并在95℃保持2分钟;
      在45分钟的时间内冷却至25℃;
      继续存储温度为4°C。
    4. RNA/DNA杂交溶液(50μM)准备使用(参见Recipes)。 该溶液可以储存在-20℃。

  2. 抑制剂稀释液的制备
    1. 将化合物的粉末溶解在DMSO中,浓度为10mM,   (例如:10",50Hz),并且如果需要(例如:2'45℃)加热它, 以确保所有化合物在溶液中
    2. 从开始 10mM的DMSO溶液使化合物在Milli-Q中的系列稀释 纯化水:从1mM稀释开始并制备三倍 相对于最终稀释10倍的浓缩物 浓度。 例如:1mM - > 333.33μM->111.11μM稀释 将导致100μM->33.33μM-> 11.11μM。 请 见注释。

  3. RNase H测定
    1. 计划没有RT酶的实验(空白),使用RT酶 缺乏抑制剂(对照),并且在存在增加的情况下 抑制剂(样品)的浓度。 最好使用较宽的范围 的浓度具有更好的抑制曲线。 反应 体积为100μl
    2. 在一个OPTIPLATE-96 F/50B中,首先加水(空白为20μl,对照为10μl)。
    3. 加入80μlRT反应混合物(1.25x)
    4. 在样品中加入10μl确定浓度的抑制剂稀释液(见图2为例)
    5. 最后向每个孔(对照和样品)中加入10μl酶(20ng HIV-1 RT)或(2nM PFV RT)。
    6.  盖上盖子,37°C孵育反应1小时   在用于微量培养板的Multi Thermo-Shaker中以200rpm
    7. 通过向每个孔中加入50μlEDTA(0.5M,pH 8.0)停止反应
    8. 在490/528nm(激发/发射波长)对Victor 3的反应进行定量。
    9.  通过从所有中减去空白的值来细化结果 并用样本报告样本中的所得值 抑制剂作为对照的百分比。
    10. 计算 插入抑制剂的IC 50值作为浓度   报告了产品信号减少50% 控制。

代表数据



图2.来自三次独立实验(生物学重复)的结果显示为对照的百分比。误差棒代表标准偏差。

笔记

  1. 该方案已应用于HIV-1和PFV RT酶。 然而,   它可以与几乎任何具有RNA酶H活性的RT酶一起使用。
  2. 我们建议使抑制剂稀释在水中,因为DMSO 溶剂影响RNase H活性,因此其在样品中的浓度 不应该克服1.01%。

食谱

  1. 反应混合物(1.25x)
    对于HIV-1 RT:
    62.5mM Tris-HCl(pH7.8)
    7.25M MgCl 2
    1.25 M DTT
    100 mM KCl
    0.3125μMRNA/DNA杂交体
  2. PFV RT反应混合物(1.25x)
    62.5mM Tris-HCl(pH8.1) 7.25mM MgCl 2
    1.25 mM DTT
    100 mM KCl
    0.3125μMRNA/DNA杂交体

致谢

带有p(His)6标签的p66/p51 HIV-1HXB2 RT-prot质粒由Stuart Le Grice Laboratory(NCI Frederick)友情提供。 泡沫病毒逆转录酶由Birgitta M.WollerUniversitätBayreuth,Lehrstuhl Biopolymere,Bayreuth,Germany提供。 该方案由Parniak等人(2003)修改和改编。

参考文献

  1. Corona,A.,Di Leva,FS,Thierry,S.,Pescatori,L.,Cuzzucoli Crucitti,G.,Subra,F.,Delelis,O.,Esposito,F.,Rigogliuso,G.,Costi, ,Cosconati,S.,Novellino,E.,Di Santo,R。和Tramontano,E。(2014a)。 Diketo酸衍生物识别参与抑制HIV-1 RNA酶H功能的高度保守的残基。/a> Antimicrob Agents Chemother 58(10):6101-6110。
  2. Corona,A.,Schneider,A.,Schweimer,K.,Rösch,P.,Wöhrl,B.M.and Tramontano,E。(2014b)。 人类免疫缺陷病毒1型核糖核酸酶H抑制剂抑制泡沫病毒逆转录酶。 em> Antimicrob Agents Chemother 58(7):4086-4093。
  3. Parniak,M.A.,Min,K.L.,Budihas,S.R.,Le Grice,S.F.and Beutler,J.A。(2003)。 基于荧光的高通量筛选测定人类免疫缺陷病毒-1逆转录酶相关的抑制剂核糖核酸酶H活性。 Anal Biochem 322(1):33-39。
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How to cite this protocol: Corona, A. and Tramontano, E. (2015). RNase H Polymerase-independent Cleavage Assay for Evaluation of RNase H Activity of Reverse Transcriptase Enzymes. Bio-protocol 5(16): e1561. DOI: 10.21769/BioProtoc.1561; Full Text



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