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Jun 2019
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Measuring Real-time DNA/RNA Nuclease Activity through Fluorescence
通过荧光测量实时 DNA/RNA 核酸酶活性   

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Abstract

DNA and RNA nucleases are wide-ranging enzymes, taking part in broad cellular processes from DNA repair to immune response control. Growing interest in the mechanisms and activities of newly discovered nucleases inspired us to share the detailed protocol of our nuclease assay (Sheppard et al., 2019). This easy and inexpensive method can provide data that enables understanding of the molecular mechanism for novel or tested nucleases, from substrate preference and cofactors involved to catalytic rate of reaction.

Keywords: Nuclease assay (核酸酶检测 ), RNA (RNA), DNA (DNA), Biochemistry (生物化学), Catalytic activity (催化活性)

Background

Nucleases are enzymes that act on DNA and RNA by cleaving the phosphodiester bonds between nucleotides. In addition to their crucial role in the DNA repair machinery, they are involved in cell signalling pathways pertinent to DNA damage and immune responses, among others (Sheppard et al., 2018). Their complex roles support several premature ageing-, immune-, and tumour-related processes. All of these can suffer from aberrations in the structural and/or catalytic functions of DNA and RNA nucleases (reviewed in Bartosova et al., 2014; Rigby et al., 2015). The growing interest in understanding the activities of numerous human DNA nucleases that remain contentious [e.g., Mre11 (Paull and Deshpande, 2014) and CTIP (Mozaffari et al., 2021)] and the presence of several uncharacterised proteins with predicted nuclease domains in mammalian genomes led us to design a real-time nuclease assay.


The activity and kinetics of nucleases, DNA polymerases, nickases, RNA:DNA nucleases, or single-strand DNA nucleases can be studied in an uncomplicated and cost-effective manner. The fluorescence signal changes resulting from decreasing amount of intercalated DNA dye can be quickly and safely measured by most plate readers. A wide range of oligonucleotides mimicking the DNA substrate of the tested enzyme can be examined in each experiment simultaneously. We designed and tested an oligomer library of 80mers with different characteristics and substrate potential. The oligonucleotides described allow for the determination of the enzymatic direction of nuclease activity. For example, 3′ or 5′ activity can be tested and compared with oligonucleotides containing biotin- blocked or free 3′ends or 5′ends, in addition to overhangs, gaps, or nicks. This method can also illustrate the importance of cofactors or cations through simple comparison between reactions supplemented or not with the chemical/cation. Using the same principle, a modified protein (phosphorylation, dephosphorylation) or mutated/truncated forms can be easily tested for their nuclease activity. Importantly, the assay is sensitive enough to detect the kinetics of repair enzymes when confronted with DNA mismatches or DNA methylation sites.

Materials and Reagents

Prepare all buffers and solutions using ultrapure, nuclease free-water and analytical grade reagents. Filter with 0.2 µm filter at least once and store at 4°C or -20°C. Always use nuclease-free tubes and cotton-filter tips.

  1. Black bottom plates [black bottom plate 96 well, polypropylene, flat bottom (Chimney well)] are necessary for the fluorescence assay to reduce background and crosstalk, and to absorb light (Greiner Bio-One, catalog number: 655209)

  2. Streptavidin (Pierce, catalog number: PIER21122)

    Streptavidin (SA) has a great affinity to biotin triethyleneglycol (BITEG). Oligonucleotides with this modification on 3′, 5′, or both ends are protected from nuclease activity after adding 2 μl (0.02 mg/ml) streptavidin to the reaction mix. Prepare 1 mg/ml in ultrapure water.

  3. Oligonucleotides

    Order lyophilised unmodified HPLC-purified oligonucleotide substrates and biotin or BITEG modified HPLC-purified oligonucleotides (Integrated DNA Technologies). Dilute them in 1× Annealing Buffer (Sigma-Aldrich) for DNA substrates (100 μM stock) and siMAXTM dilution buffer (Eurofins; 30 mM HEPES, 100 mM KCl, 1 mM MgCl2, pH = 7.3) for all RNA substrates.

    Note: Modified DNA or RNA bases can be ordered in the synthesised oligos. We have done so successfully in the past to measure the effect of base modifications on enzymatic functions.


    Oligonucleotides from our library were designed and optimised against secondary structure formation using the ‘Predict a Secondary Structure Web Server’ (https://rna.urmc.rochester.edu/RNAstructureWeb/Servers/Predict1/Predict1.html).

    The basic 80bp sequences are as follows:

    Forward: GATGGTTTGTTGGTCTATTACTACTTGGAGCTTGTATGATTCGAAACCTTGGAGTACTTGCCTACTTGGAGTGAACTTAG

    Reverse: CTAAGTTCACTCCAAGTAGGCAAGTACTCCAAGGTTTCGAATCATACAAGCTCCAAGTAGTAATAGACCAACAAACCATC


    For designing all types of library oligomers, the basic oligonucleotide sequence can be shortened from both ends or split into two molecules to produce gaps or nicks in duplex DNA. Single nucleotides can be exchanged to add mismatches, substrate preferences, and so forth (Figures 1 and 2).



    Figure 1. For the calibration curve, two example sets are presented, with and without streptavidin blockade on ends. These need to match the DNA substrates used in the experiment.



    Figure 2. Set of 80-mer oligonucleotide substrates


  4. Nucleases

    Perform the calibration of the method using the commercial nucleases with known activity and preferably acting in a similar mechanism to the predicted activity of the enzyme to be tested. For example, to test the removal of 5′ mononucleotides from duplex DNA, use Exonuclease T7 (T7, New England Biolabs, catalog number: M0263S). For nucleases acting 3′ to 5′, use Exonuclease III (ExoIII, Thermo Scientific, catalog number: EN0191) (Figure 3).



    Figure 3. Scheme of PG release from dsDNA after nuclease treatment (ExoIII)


  5. 1 M Tris, pH 7.5-8.0 (ThermoScientific, catalog number: 15567027)

  6. 5 M NaCl, RNase-free (ThermoScientific, catalog number: AM9759)

  7. 0.5 M EDTA pH 8.0 (ThermoScientific, catalog number: 15575020)

  8. 10× Tango Buffer (ThermoScientific, catalog number: BY5)

  9. CutSmart (New England Biolabs, catalog number: B7204S)

  10. Glycerol (ThermoScientific, catalog number: 15514011)

  11. Quant-iTTM PicoGreenTM dsDNA Assay Kit (Invitrogen, catalog number: P7589)

  12. Quant-iTTM microRNA Assay Kit (Invitrogen, catalog number: Q33140)

  13. Annealing Buffer Composition (1×) (see Recipes)

  14. Storage buffers (see Recipes)

  15. Reaction buffers (see Recipes)

  16. Divalent cations (see Recipes)

  17. PG buffer (see Recipes)

  18. Nucleic acid dyes (see Recipes)

Equipment

  1. Plate reader (Tecan or BMG)

    Select the appropriate measurement parameters (temperature, wavelength, number of flashes, and settle time) for your assay.

      Preheat the microplate reader to the optimal temperature for enzyme activity (commonly 37°C). To slow down the reaction, set to a lower temperature (e.g., 20°C). Use the wavelength 483-15 nm for excitation (483 nm is the middle excitation peak with a bandwidth of 15 nm; i.e., the excitation range is 475-490 nm) and 530-30 nm for emission (530 nm is the middle emission peak with a bandwidth of 30 nm; i.e., the emission range is 515-545 nm). Increase the number of flashes per well until noise of BLANK wells does not improve further or until measurement time per well becomes unacceptable. Depending on the enzyme, read the samples every 45-60 s for 15 min to 2 h with a shake before each read.

      Photobleaching occurring in the samples causes the fluorescence signal to decrease with time. Therefore, run a control curve run in every experiment simultaneously with samples to measure this effect. Longer readings can be inaccurate due to total photobleaching of PicoGreen to the level of the background.

      We tested Infinite M Plex (TECAN) and CLARIOstar (BMG Labtech) plate readers. The nuclease assay can also be performed in the qPCR reader, but we found that the plate reader gives consistent results and offers more options, such as shaking the plate before a read.

      While the best practices regarding reads per well vary with each enzyme, in general, an optimal duration of reads is approximately 1 h. If using a CLARIOstar, factor the whole time needed to read the entire plate, well by well, which for a full plate took approximately 48 s. So, each well could only be read every 50 s. If the plate reader permits it, try to use bidirectional reading row by row, and add the enzymes in the same order.

Procedure

  1. Plate Preparation

    Prepare the plate on ice and protect the samples from light. For every reaction, prepare the mix and run triplicates for the best accuracy. Use of multi-channel pipettes accelerates the set up of the reaction plate but can also lead to air bubbles in wells. Centrifuge the plate to remove them.

    Always mark up the plate to aid with pipetting substrate and reaction mixtures and to blank off any empty wells. If using tape to label the rows and columns, peel this off before loading into the plate reader. Take time to plan out experiments and calculate volumes well ahead of time.


    Reaction mix per well:

    10 µl oligonucleotides 500 nM

    10 µl reaction buffer

    25 µl ddH2O (or 23 µl if SA)

    2 µl of streptavidin (for biotinylated oligos)

    50 µl PG

    5 µl enzyme

    Note: For RNA reactions, a similar procedure could be followed by substituting PG with RNA dye.


    1. Perform the annealing of complementary oligonucleotides in a thermocycler or heat block and use the molar ratio 1:1.

      1. Mix equal volumes of the equimolar oligonucleotides (100 µM each) in a PCR tube.

      2. Incubate for 5 min at 95°C.

      3. Switch off the heat block leaving the samples in to cool down slowly or use the thermocycler (go down 1°C every 30 s).

      4. Keep the annealed duplexes at -20°C for long-term storage.

      5. Dilute the annealed oligos to 500 nM.

      6. Use 10 µl (50 nM) per reaction/well.

        For example, to anneal the oligonucleotide duplexes containing the gap, anneal three oligonucleotides using 2.5 µl each, and add 2.5 µl of annealing buffer. The concentration of the end product is 25 µM.

        Notes:

        1. Use the reaction buffer appropriate for the enzyme. The wide range of activity buffers, like 10× CutSmart (New England Biolabs, B7204S) or 10× Tango (ThermoScientific, BY5), can facilitate the data analysis (no need for extra controls) and work well with many enzymes. Run a trial experiment to test the effectiveness of different reaction buffers to get the best results.

        2. Use nuclease-free water.

    2. Remember to add 2 µl of streptavidin for BIOTEG or biotinylated oligonucleotides. Incubate for 15 min on ice on a see-saw shaker to enhance binding.

    3. Add PicoGreen reagent (PG) to every well protecting the plate from light. Try to work quickly and cover the rows/columns already supplemented with PG with lid/aluminium foil.

    4. Add the denoted enzymes (or storage buffer as a negative control) to each well.

      Work at the bench at room temperature from now on to avoid the risk of enzyme precipitation. Prepare the desired amount of enzyme units/concentration in 5 µl. Take a full box of filter tips and use in the corresponding position in the tip box and plate to avoid pipetting mistakes. This way, if the position on the plate is lost, the tip box could orient the processing. Add 5 µl to each well, trying not to make any bubbles that could affect the fluorescence reads. The reaction starts now, so work quickly to be able to catch the first minutes of the reaction in the pre-heated plate reader. Remember to protect the plate from light.

      Notes:

      1. Avoid using multi-channel pipettes as they tend to add bubbles to wells and are less consistent in adding the correct amount of enzyme.

      2. Centrifuge the plate briefly to remove air bubbles. For quick acting enzymes, this may not be advisable as the enzymatic reaction may have already started during that time.

      3. Attempt to titrate out the enzyme. If too much enzyme is present in the solution, the reaction is very fast, causing loss in data read in the early phase of the reaction.

    1. Design of experiment

      Plate with test experiment with T7 and ExoIII exonucleases. The samples were prepared in duplicate. The calibration control curve is also included, as illustrated in Figure 4.



      Figure 4. Scheme of the plate for test experiment. A. Row A1-6 contains the negative control oligomer for both tested exonucleases. The correct binding between biotin-modified-oligonucleotides and streptavidin added to the mix protects both 3′ and 5′ ends from digestion. All4block signifies that all four ends on the oligos were blocked by biotin-streptavidin binding. B. Row B1-6 contains oligonucleotides with 3bp gap in proximity, 12bp to 3′ end (which is the substrate for ExoIII acting in the 3′ to 5′ direction). T7 could only digest short 12bp fragments leading to the last nucleotide blocked by Biotin-Streptavidin. C. Row C1-6 contains oligonucleotides with 3bp gap in proximity, 12bp to 5′ end (which is the substrate for T7 exonuclease acting in the 5′ to 3′ direction). ExoIII could only digest short 12bp fragments leading to the last nucleotide blocked by Biotin-Streptavidin. D. Row D1-6 contains oligonucleotides with 15bp overhang on 5′. E. Row E1-6 contains oligonucleotides with 15bp overhang on 3′. In Columns 1 and 2, storage buffer from ExoIII (or T7) was added to the samples. Columns 3 and 4: ExoIII 5U per well. Columns 5 and 6: T7 5U per well. A7 and A8: 80mer_3block (oligos that 80mers with both 3′ ends blocked). B7 and B8: 40mer_3block (oligos that 40mers with both 3′ ends blocked). C7 and C8: ssDNA, 80bp single strain DNA, as a control of totally digested duplex oligomer, minimum value of fluorescence for the experiment.

    Data analysis

    1. Create the calibration lines from the oligonucleotides with different nominal lengths: 80, 40, and 0 (single stranded). Obtain the reads from the oligonucleotides as a function of time, possibly two or three replicates per nominal length. Next, at each time point, calculate the linear relationship between the nominal lengths (80, 40, 0) and the raw fluorescence reads. As a result, a time evolution of slopes and intercepts that relate the control signal to the control nominal length is obtained.

    2. Group the data reads of the same oligonucleotide in sets with appropriate negative control, which must be very similar to the tested sample. It could contain no enzyme, or optimally, the inactive form of the tested enzyme. DNA 80-oligomer, which is not the substrate for the enzyme, i.e., 80 mer_all4block, can be the negative control too. At the zero-time point, the raw reads should be similar for all wells.

    3. Correct for the effects of photobleaching and background reads by means of dividing the reads of the specimen by the average reads from the negative control well.

    4. Using the slope and intercept from calibration lines, convert the corrected fluorescence units at each time point to length of duplex DNA. See Supplementary material (Excel file with test experiment and calculations) for a template of data entry and calculations.

    5. The result should reflect the length of oligomers, where 80mer is the maximum size, and single ssDNA shows total dissolved duplex.

      Both nucleases were tested on five different oligomers from the library.

      The 80mer-all4block duplex is not the substrate for any of the tested nucleases (no nickase activity). Thus, we used the 80mer-all4block as a negative control in calculations. The duplex cannot be degraded during the assay because changes in fluorescence occur only due to photobleaching of the PG reagent.

      1. ExoIII activity nuclease assay

        ExoIII, with its 3' to 5' exodeoxyribonuclease activity, releases 5'-mononucleotides from the 3'-end. It acts effectively on 80 mer-3block-5′-15 bp_overhang and on 80mer-all4block-3bpgap-3′.

        80mer-all4block-3bpgap-5′ has a gap situated 12bp from the 5′ end of the oligomer. The decrease in fluorescence signal reflects the ability of ExoIII to remove this few bp from the 3′-end starting in the gap.

        The lower 80mer_all4block control fluorescence signal compared to 80mer-3block-3′-15bp_overhang confirms that the PG reagent intercalates only with double stranded DNA and not with the ssDNA overhang that is part of the oligo and not a substrate for ExoIII.

        The reaction with ExoIII starts quickly, and in this experiment, the substrate oligomers for ExoIII were registered at time point 0 as 60bp and 50bp fragments, respectively (Figure 5A).

      2. T7 activity nuclease assay

        T7 exonuclease with its 5′ to 3' activity, non-processively hydrolyses oligomers starting on free 5′-end on both 80mer-3block-3′-15bp and 80mer-all4block-3bpgap-5′. T7 can also remove few bp from the 5′-end of the gap in 80 mer-all4block-3bpgap-5′ (Figure 5B).



      Figure 5. Sample of the nuclease activity results. A. ExoIII nuclease activity, 5U per well. B. T7 nuclease activity, 5U per well. The graphs from the test experiment with ExoIII exonuclease and T7 exonuclease. The data is presented for each enzyme separately. The 80bp oligomer and ssDNA are shown on the graph. The x-axis denotes time in seconds. The y-axis denotes DNA oligo length in base pairs.


    Summary: The nuclease assay summarised in this protocol is a powerful and easy toolkit for analysis of the activity of enzymes connected to DNA/RNA. Wide application, simplicity of test preparation, affordable reagents, and accessible equipment makes it a great method to implement in every molecular biology lab. We also postulate that this assay could be used in various variations to measure nucleic acid metabolism through non-nuclease activities.

    Recipes

    1. Annealing Buffer Composition (1×)

      Make 100 µM stock of the oligonucleotides (ordered from IDT [idtdna.com])

      10 mM Tris, pH 7.5-8.0

      50 mM NaCl

      1 mM EDTA pH 8.0

    2. Storage buffers

      Prepare enzymes’ specific storage buffers for both control wells (without any enzyme) and for diluting the enzyme. Do not add glycerol.

      - For ExoIII enzyme: 50 mM Tris-HCl (pH 8.0), 50 mM KCl, 1 mM DTT (add fresh prior to use).

      - For T7 enzyme: 10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, 10 mM DTT (add fresh prior to use).

      Note: These storage buffers vary depending on the specific enzyme and the manufacturer producing it. They are usually provided by the manufacturer when purchasing specific enzymes.

    3. Reaction buffers

      Use recommend using reaction buffers delivered with nucleases. For unknown preference of tested nucleases, try wide range activity buffers, i.e., 10× Tango Buffer and 10× CutSmart.

      - 10× Reaction Buffer for ExoIII: 660 mM Tris-HCl (pH 8.0 at 30°C), 6.6 mM MgCl2.

      - 10× Reaction Buffer T7: 10× NEBuffer 4 (B7004S): 500 mM potassium acetate, 200 mM Tris acetate, 100 mM magnesium acetate, 10 mM DTT, pH 7.9 at 25°C.

      Note: As reaction buffers can make a big difference, research the best one for each enzyme carefully and use the most appropriate one for the specific enzyme being tested.

    4. Divalent cations

      Supplement reaction buffer with MgCl2 or MnCl2 when needed. Use both in the first experiments; the information about preferred cation can be determined later.

    5. PG buffer

      TE buffer with v/v glycerol for diluting PG reagent

      10 mM Tris-HCl

      1 mM EDTA, pH 7.5

      40% glycerol

    6. Nucleic acid dyes

      DNA dyes: The PicoGreen (PG) reagent from Quant-iTTM PicoGreenTM dsDNA Assay Kit was prepared immediately (producer recommendation) before use by making a 1:200 dilution of the PG in TE buffer with v/v glycerol.

      Store the PG reagent long term at -20°C; while thawing, wipe off any moisture before opening the tube. Light and moisture both harm the PG reagent. We routinely keep the PG aliquoted and frozen, 5 µl per tube (1 ml of PG Buffer to be added prior to use), in a light proof box.

      Note (RNA dyes): For the RNase assays, the dye from the Quant-iTTM microRNA Assay Kit was prepared by diluting the microRNA reagent A into buffer B in a 1/2,000 dilution, as detailed in the protocol.

    Acknowledgments

    The RC lab is funded by the BBSRC (BB/N017773/2), SNF (CRSK-3_190550), Rosetrees Trust Fund (M713), and UZH Research Priority Program (URPP–Translational Cancer Research). This protocol was adapted from the publication by Sheppard et al. (2019).

    Competing interests

    The authors declare no competing interests.

    References

    1. Bartosova, Z. and Krejci, L. (2014). Nucleases in homologous recombination as targets for cancer therapy. FEBS Lett 588(15): 2446-2456.
    2. Mozaffari, N. L., Pagliarulo, F. and Sartori, A. A. (2021). Human CtIP: A 'double agent' in DNA repair and tumorigenesis. Semin Cell Dev Biol 113: 47-56.
    3. Paull, T. T. and Deshpande, R. A. (2014). The Mre11/Rad50/Nbs1 complex: recent insights into catalytic activities and ATP-driven conformational changes. Exp Cell Res 329(1): 139-147.
    4. Rigby, R. E. and Rehwinkel, J. (2015). RNA degradation in antiviral immunity and autoimmunity. Trends Immunol 36(3): 179-188.
    5. Sheppard, E. C., Morrish, R. B., Dillon, M. J., Leyland, R. and Chahwan, R. (2018). Epigenomic modifications mediating antibody maturation. Front Immunol 9: 355.
    6. Sheppard, E. C., Rogers, S., Harmer, N. J. and Chahwan, R. (2019). A universal fluorescence-based toolkit for real-time quantification of DNA and RNA nuclease activity. Sci Rep 9(1): 8853.

简介

[摘要] DNA 和 RNA 核酸酶是范围广泛的酶,参与广泛的细胞过程从 DNA 修复到免疫反应控制。对新的机制和活动越来越感兴趣发现的核酸酶启发我们分享核酸酶测定的详细方案(Sheppard等人,2019)。这种简单且廉价的方法可以提供有助于理解分子结构的数据。新的或经过测试的核酸酶的机制,从底物偏好和辅助因子到催化反应速率。

[背景]核酸酶是通过裂解磷酸二酯键作用于 DNA 和 RNA 的酶核苷酸之间。除了它们在 DNA 修复机制中的关键作用外,它们还参与与 DNA 损伤和免疫反应等相关的细胞信号通路(Sheppard等阿尔。, 2018)。它们的复杂作用支持多种过早衰老、免疫和肿瘤相关过程。所有这些都可能遭受 DNA 结构和/或催化功能的畸变和RNA 核酸酶(Bartosova等人综述,2014 年;Rigby等人,2015 年)。日益增长的兴趣了解许多仍有争议的人类 DNA 核酸酶的活性 [例如,Mre11(Paull and Deshpande, 2014) 和 CTIP (Mozaffari et al ., 2021)] 以及几个哺乳动物基因组中具有预测核酸酶结构域的未表征蛋白质使我们设计了一个实时核酸酶检测。核酸酶、DNA 聚合酶、切口酶、RNA:DNA 核酸酶或单链酶的活性和动力学可以以简单且经济高效的方式研究链 DNA 核酸酶。荧光由于插入的 DNA 染料量减少而导致的信号变化可以快速安全地由大多数读板机测量。模拟 DNA 底物的多种寡核苷酸测试酶可以在每个实验中同时检测。我们设计并测试了一个具有不同特性和底物潜力的 80 聚体寡聚体文库。寡核苷酸所描述的允许确定核酸酶活性的酶促方向。例如,3′或 5' 活性可以测试并与含有生物素封闭或游离 3' 端的寡核苷酸进行比较或 5' 端,以及突出、间隙或缺口。这个方法也可以说明通过简单比较补充或不补充的反应之间的辅因子或阳离子化学/阳离子。使用相同的原理,修饰的蛋白质(磷酸化、去磷酸化)或可以很容易地测试突变/截短形式的核酸酶活性。重要的是,该测定是当遇到 DNA 错配或DNA 甲基化位点。

关键字:核酸酶检测 , RNA, DNA, 生物化学, 催化活性

材料和试剂

使用超纯、无核酸酶水和分析级试剂制备所有缓冲液和溶液。

至少用 0.2 µm 过滤器过滤一次,并在 4°C -20°C 下储存。始终使用无核酸酶管和棉花过滤器技巧。

1.黑色底板【黑色底板96孔,聚丙烯,平底(烟囱孔)】是减少背景和串扰以及吸收光的荧光分析所必需的(Greiner Bio-One,目录号:655209。)

2.链霉亲和素(Pierce,目录号:PIER21122)链霉亲和素 (SA) 对生物素三甘醇 (BITEG) 具有很强的亲和力。寡核苷酸与添加 2 μl 后,3'5' 或两端的这种修饰不受核酸酶活性的影响(0.02 mg/ml) 链霉亲和素加入到反应混合物中。在超纯水中制备 1 mg/ml

3.寡核苷酸订购冻干的未改性 HPLC 纯化的寡核苷酸底物和生物素或 BITEG改良的 HPLC 纯化寡核苷酸(集成 DNA 技术)。将它们稀释1 倍用于 DNA 底物的退火缓冲液 (Sigma-Aldrich)100 μM 原液)和 siMAX TM稀释缓冲液(Eurofins; 30 mM HEPES, 100 mM KCl, 1 mM MgCl 2 , pH = 7.3) 适用于所有 RNA 底物。

注意:可以在合成的寡核苷酸中订购修饰的 DNA RNA 碱基。我们这样做了过去成功地测量碱基修饰对酶促功能的影响。

我们文库中的寡核苷酸是针对二级结构进行设计和优化的形成使用这'预测一种中学结构网络服务器'

https://rna.urmc.rochester.edu/RNAstructureWeb/Servers/Predict1/Predict1.html )

基本的80bp序列如下:

向前:

GATGGTTTGTTGGTCTATTACTACTTGGAGCTTGTATGATTCGAAACCTTGGAGTACTTGCC

TACTTGGAGTGAACTTAG

逆转:

CTAAGTTCACTCCAAGTAGGCAAGTACTCCAAGGTTTCGAATCATACAAGCTCCAAGTAGT

AATAGACCAACAAACCATC

对于设计所有类型的文库寡聚体,基本寡核苷酸序列可以是从两端缩短或分裂成两个分子以在双链 DNA 中产生间隙或切口。可以交换单个核苷酸以添加错配、底物偏好等(图 1 和图 2)。

1. 对于校准曲线,提供了两个示例集,有和没有链霉亲和素阻断末端。这些需要与用于检测的 DNA 底物相匹配。实验

2. 一组 80 聚体寡核苷酸底物

4.核酸酶使用已知活性的商业核酸酶对方法进行校准,并且优选以与待测酶的预测活性相似的机制起作用。例如,要测试从双链 DNA 中去除 5' 单核苷酸,请使用核酸外切酶 T7T7,新英格兰生物实验室,目录号:M0263S)。对于作用于 3' 5' 的核酸酶,使用核酸外切酶 IIIExoIIIThermo Scientific,目录号:EN0191)(图 3)。

3. 核酸酶处理后从 dsDNA 中释放 PG 的方案 (ExoIII)

5. 1 M TrispH 7.5-8.0ThermoScientific,目录号:15567027

6. 5 M NaCl,无RNaseThermoScientific,目录号:AM9759

7. 0.5 M EDTA pH 8.0ThermoScientific,目录号:15575020

8. 10× Tango BufferThermoScientific,目录号:BY5

9. CutSmart(新英格兰生物实验室,目录号:B7204S

10.甘油(ThermoScientific,目录号:15514011

11. Quant-iT TM PicoGreen TM dsDNA 检测试剂盒(Invitrogen,目录号:P7589

12. Quant-iT TM microRNA 检测试剂盒(Invitrogen,目录号:Q33140

13.退火缓冲液组合物()(见配方)

14.存储缓冲区(见食谱)

15.反应缓冲液(见配方)

16.二价阳离子(见配方)

17. PG 缓冲液(见配方)

18.核酸染料(见配方)

设备

1.        读板机(Tecan BMG

选择合适的测量参数(温度、波长、闪光次数、和稳定时间)用于您的检测。将酶标仪预热至酶活性的最佳温度(通常为37°C)。要减慢反应速度,请设置较低的温度(例如20°C)。使用波长483-15 nm 用于激发(483 nm 是中间激发峰,带宽为 15 nm;即激发范围为 475-490 nm)和 530-30 nm 的发射(530 nm 是中间发射峰值带宽为 30 nm;即,发射范围为 515-545 nm)。增加数量每孔闪烁直到空白孔的噪音没有进一步改善或直到测量每口井的时间变得不可接受。根据酶的不同,每 45- 读取一次样品每次读取前摇晃 60 秒,持续 15 分钟至 2 小时。样品中发生的光漂白导致荧光信号随着时间。因此,在每个实验中与样品同时运行控制曲线测量这种效果。由于完全光漂白,较长的读数可能不准确PicoGreen 到背景的水平。

我们测试了无限 M Plex (TECAN) CLARIOstar (BMG Labtech) 读板机。这核酸酶检测也可以在 qPCR 阅读器中进行,但我们发现酶标仪提供一致的结果并提供更多选项,例如在读取前摇动板。

虽然关于每孔读数的最佳实践因每种酶而异,但一般而言,读取的最佳持续时间约为 1 小时。如果使用 CLARIOstar,请考虑整个时间需要逐个读取整个板,对于整个板大约需要 48 秒。所以,每个孔只能每 50 秒读取一次。如果读板器允许,请尝试使用双向逐行阅读,并按相同顺序添加酶。

程序

A. 板准备

在冰上准备板并保护样品免受光照。对于每个反应,准备混合物和运行三次以获得最佳准确度。使用多通道移液器可加快设置反应板,但也可能导致孔中出现气泡。离心板以去除它们。始终标记板以帮助移取底物和反应混合物并清除任何空井。如果使用胶带标记行和列,请在装入盘子之前将其撕下读者。花时间计划实验并提前计算体积。每孔反应混合物:

10 µl 寡核苷酸 500 nM

10 微升反应缓冲液

25 µl ddH 2 O(如果是 SA,则为 23 µl

2 µl 链霉亲和素(用于生物素化寡核苷酸)

50微升PG

5微升酶

注意:对于 RNA 反应,可以遵循类似的程序,用 RNA 染料代替 PG

1. 在热循环仪或加热块中对互补寡核苷酸进行退火,然后使用摩尔比 1:1。一种。在 PCR 管中混合等体积的等摩尔寡核苷酸 (每个 100 µM)。湾  95°C 下孵育 5 分钟。

C。关闭加热块,让样品缓慢冷却或使用热循环仪(每 30 秒降低 1°C)。

d. 将退火双链体保持在 -20°C 以进行长期储存。

e. 将退火的寡核苷酸稀释到 500 nM

F。每个反应/孔使用 10 µl (50 nM)

例如,要退火包含缺口的寡核苷酸双链体,退火三个寡核苷酸各使用 2.5 µl,并添加 2.5 µl 退火缓冲液。的浓度最终产物为 25 µM

笔记:

一种。使用适合酶的反应缓冲液。广泛的活动缓冲区,像 10 × CutSmart (New England Biolabs, B7204S) 10 × Tango (ThermoScientific,BY5),可以方便数据分析(不需要额外的控制),并与许多酶。进行试验实验以测试不同反应的有效性缓冲以获得最佳结果。湾使用无核酸酶的水。

2. 记住为 BIOTEG 或生物素化寡核苷酸添加 2 µl 链霉亲和素。孵化为在跷跷板摇床上冰上 15 分钟以增强结合。

3. PicoGreen 试剂 (PG) 添加到保护板避光的每个孔中。尝试快速工作并用盖子/铝箔覆盖已经补充了 PG 的行/列。

4. 将指定的酶(或储存缓冲液作为阴性对照)添加到每个孔中。从现在开始在室温下在工作台上工作,以避免酶沉淀的风险。

5 µl 中制备所需数量的酶单位/浓度。带上一整盒过滤嘴并在吸头盒和板的相应位置使用,以避免移液错误。这个这样,如果板上的位置丢失,尖端盒可以定位加工。各加 5 µl好吧,尽量不要产生任何可能影响荧光读数的气泡。反应开始现在,所以快速工作,以便能够捕捉到预热板中反应的最初几分钟读者。记住要保护盘子免受光照。

笔记:一种。避免使用多道移液器,因为它们往往会向孔中添加气泡,并且较少加入正确量的酶是一致的。湾 将板短暂离心以除去气泡。对于速效酶,这可能不会由于酶促反应可能在此期间已经开始,因此是可取的。

C。尝试滴定酶。如果溶液中存在过多的酶,则反应

速度非常快,导致反应早期读取的数据丢失。

B. 实验设计

带有 T7 ExoIII 核酸外切酶测试实验的平板。样品制备于复制。还包括校准控制曲线,如图 4 所示。

4. 用于测试实验的板方案。A. A1-6 行包含阴性对照两种测试的核酸外切酶的寡聚体。生物素修饰-之间的正确结合添加到混合物中的寡核苷酸和链霉亲和素可保护 3' 5' 末端不被消化。All4block 表示寡核苷酸上的所有四个末端都被生物素-链霉亲和素结合阻断。B. B1-6 行包含在 12bp 3' 末端(这是ExoIII 作用于 3' 5' 方向的底物)。T7 只能消化 12bp 的短片段导致最后一个核苷酸被生物素-链霉亲和素阻断。C. C1-6 行包含接近 3bp 间隙的寡核苷酸,从 12bp 5' 末端(这是 T7 的底物)作用于 5' 3' 方向的外切核酸酶)。ExoIII 只能消化 12bp 的短片段导致最后一个核苷酸被生物素-链霉亲和素阻断。D. D1-6 行包含在 5' 上具有 15bp 突出端的寡核苷酸。E. E1-6 行含有 15bp 的寡核苷酸悬垂在 3'。在第 1 列和第 2 列中,将来自 ExoIII(或 T7)的存储缓冲液添加到样品中。第 3 和第 4 列:每孔 ExoIII 5U。第 5 和第 6 列:每孔 T7 5UA7 A880mer_3block(具有两个 3' 末端的 80 聚体被阻断的寡核苷酸)。B7 B840mer_3block40mers 的寡核苷酸)3' 端都被封闭)。C7 C8ssDNA80bp 单株 DNA,作为完全对照消化的双链寡聚体,实验的荧光最小值。

数据分析

1.从具有不同标称长度的寡核苷酸创建校准线:8040 0(单股)。获取作为时间函数的寡核苷酸读数,可能每个标称长度两个或三个重复。接下来,在每个时间点,计算线性标称长度 (80, 40, 0) 和原始荧光读数之间的关系。其结果,将控制信号与控制标称相关联的斜率和截距的时间演变得到长度。

2.将相同寡核苷酸的数据读数与适当的阴性对照分组,这必须与测试样品非常相似。它可以不含酶,或者最好是被测酶的非活性形式。DNA 80-寡聚体,它不是酶的底物,即., 80 mer_all4block,也可以是阴性对照。在零时间点,原始读数所有井都应该是相似的。

3.通过划分reads,修正光漂白和背景reads的影响样本的平均读数来自阴性对照孔。

4.使用校准线的斜率和截距,将校正后的荧光单位转换为每次都指向双链 DNA 的长度。见补充材料(带测试的 Excel 文件实验和计算)用于数据输入和计算的模板。

5.结果应反映低聚物的长度,其中 80mer 为最大尺寸,单ssDNA 显示总溶解双链体。乙OTH核酸酶从文库五个不同的低聚物进行测试。80mer-all4block 双链体不是任何测试核酸酶的底物(无切口酶活动)。因此,我们在计算中使用 80mer-all4block 作为阴性对照。复式不能在测定过程中,因为通道被降解安格斯在荧光只发生因PG 试剂的光漂白。

一种。ExoIII 活性核酸酶测定ExoIII 具有3' 5' 脱氧核糖核酸酶活性,从3'端。它对 80 mer-3block-5′-15 bp_overhang 80mer-all4block- 有效3bpgap-3'80mer-all4block-3bpgap-5' 在距离寡聚体 5' 12bp 处有一个缺口。这荧光信号的降低反映了 ExoIII 3'-端从间隙开始。与 80mer-3block-3'- 相比,较低的 80mer_all4block 对照荧光信号15bp_overhang 确认 PG 试剂仅插入双链 DNA而不是与作为寡核苷酸一部分而不是 ExoIII 底物的 ssDNA 悬垂。与 ExoIII 的反应很快开始,在这个实验中,底物低聚物为ExoIII 在时间点 0 分别注册为 60bp 50bp 片段(图 5A)。湾 T7活性核酸酶测定具有 5' 3' 活性的 T7 核酸外切酶非持续性地水解寡聚体80mer-3block-3'-15bp 80mer-all4block-3bpgap-5' 上的游离 5'-末端。T7还可以从 80 mer-all4block-3bpgap-5' 间隙的 5' 端去除几个 bp(图 5B)。

5.核酸酶活性结果的样本。A. ExoIII 核酸酶活性,每孔 5UB.T7 核酸酶活性,每孔 5U。来自 ExoIII 核酸外切酶测试实验的图表和 T7 核酸外切酶。分别提供每种酶的数据。80bp 寡聚体和 ssDNA 显示在图表上。x 轴表示以秒为单位的时间。y 轴表示碱基对中的 DNA 寡核苷酸长度。

总结:本协议中总结的核酸酶检测是一个功能强大且简单的工具包,可用于分析与 DNA/RNA 相关的酶的活性。应用广泛,测试简单准备、负担得起的试剂和易于使用的设备使其成为实施的好方法在每个分子生物学实验室。我们还假设该测定可用于各种变化通过非核酸酶活性测量核酸代谢。

食谱

1. 退火缓冲液组成(

制备 100 µM 寡核苷酸库存(从 IDT [idtdna.com] 订购)

10 mM TrispH 7.5-8.0

50 毫米氯化钠

1 mM EDTA pH 8.0

2. 存储缓冲区

为对照孔(不含任何酶)和

稀释酶。不要添加甘油。

- 对于 ExoIII 酶:50 mM Tris-HCl (pH 8.0)50 mM KCl1 mM DTT(使用前添加新鲜)。

- 对于 T7 酶:10 mM Tris-HCl (pH 8.0)0.1 mM EDTA10 mM DTT(使用前添加新鲜的)。

注意:这些储存缓冲液因特定酶和制造商而异

生产它。它们通常由制造商在购买特定酶时提供。

3. 反应缓冲液

使用建议使用随核酸酶一起提供的反应缓冲液。对于未知的偏好测试过的核酸酶,尝试使用范围广泛的活性缓冲液,10× Tango Buffer 10× CutSmart

- ExoIII 10× 反应缓冲液:660 mM Tris-HClpH 8.030°C),6.6 mM MgCl2

- 10 × 反应缓冲液 T710 × NEBuffer 4 (B7004S)500 mM 醋酸钾,200 mM Tris

醋酸盐,100 mM 醋酸镁,10 mM DTTpH 7.925°C

注意:由于反应缓冲液可以产生很大的不同,研究每种酶的最佳缓冲液

仔细并使用最适合被测试的特定酶的酶。

4. 二价阳离子

需要时用 MgCl 2 MnCl 2补充反应缓冲液。在第一个实验中使用两者;有关优选阳离子的信息可以稍后确定。

5PG缓冲液

v/v 甘油的 TE 缓冲液,用于稀释 PG 试剂

10 mM Tris-HCl

1 mM EDTApH 7.5

40% 甘油

6. 核酸染料

DNA 染料:来自 Quant-iT TM PicoGreen TM dsDNA Assay Kit PicoGreen (PG) 试剂是

使用前立即制备(生产商推荐),通过 1:200 稀释

v/v 甘油的 TE 缓冲液中的 PG

PG试剂在-20°C长期保存;解冻时,打开前擦去水分

管。光和湿气都会损害 PG 试剂。我们通常将 PG 等分并

冷冻,每管 5 µl(使用前加入 1 ml PG 缓冲液),置于避光盒中。

注(RNA 染料):对于 RNase 检测,来自 Quant-iT TM microRNA 检测试剂盒的染料通过将 microRNA 试剂 A 稀释到缓冲液 B 中以 1/2,000 稀释来制备,详见在协议中。

致谢

RC 实验室由 BBSRC (BB/N017773/2)SNF (CRSK-3_190550)Rosetrees Trust 资助Fund (M713) UZH 研究优先计划(URPP-转化癌症研究)。这个协议改编自 Sheppard等人的出版物(2019)

利益争夺

作者声明没有竞争利益。

参考

1. Bartosova, Z. Krejci, L. (2014) 同源重组中的核酸酶作为癌症靶点治疗。F EBS Lett 588(15): 2446-2456

2. Mozaffari, NL, Pagliarulo, F. Sartori, AA (2021)人类 CtIPDNA 中的双重代理修复和肿瘤发生。 精细胞开发生物学11347-56

3. Paull, TT Deshpande, RA (2014)Mre11/Rad50/Nbs1 复合体:最近对催化活性和 ATP 驱动的构象变化。Exp Cell Res 329(1): 139-147

4. Rigby, RE Rehwinkel, J. (2015)抗病毒免疫和自身免疫中的 RNA 降解。趋势免疫学363):179-188

5. Sheppard, EC, Morrish, RB, Dillon, MJ, Leyland, R. Chahwan, R. (2018) 表观基因组学介导抗体成熟的修饰。前免疫学9355

6. Sheppard, EC, Rogers, S., Harmer, NJ Chahwan, R. (2019) 通用荧光-基于工具包,用于实时定量 DNA RNA 核酸酶活性。科学报告9(1): 8853

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引用:Wyrzykowska, P., Rogers, S. and Chahwan, R. (2021). Measuring Real-time DNA/RNA Nuclease Activity through Fluorescence. Bio-protocol 11(21): e4206. DOI: 10.21769/BioProtoc.4206.
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