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Random DNA Binding Selection Assay (RDSA)
随机DNA结合选择分析(RDSA)   

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Abstract

Protein-DNA interaction is a very important cellular process, by which regulation of DNA biological function, usually gene expression, is exerted. The method of random DNA binding selection assay (RDSA) can be used to identify DNA elements bound by proteins with DNA-binding activities. This method is based on the enrichment of the target DNA element by the immobilized recombinant protein on special beads and repeated PCR amplification.

Keywords: Protein-DNA interaction(蛋白质- DNA的相互作用), Transcription factor(转录因子), Cis-element(顺式元件), Trans-element(跨元素), DNA-binding activities(DNA结合活性)

Materials and Reagents

  1. The prokaryotic expression vectors: pGEX-6P-1 (GE Healthcare) or pTXB3 (New England Biolabs)
  2. The expression strain [Escherichia coli (E.coli) BL21 (DE3) or others]
  3. T-Vector pGEMT-easy and T4-DNA ligase (Promega Corporation, catalog number: A1360 )
  4. DNA polymerase (Takara Bio Company, catalog number: DR001 )
  5. Sterile ddH2O (double distilled water)
  6. Oligonucleotides (Pitzschke et al., 2009; Wang et al., 2014)
  7. Chitin resin (New England Biolabs, catalog number: S6651S )
  8. PBS buffer (see Recipes)
  9. Column buffer (see Recipes)
  10. RDSA buffer (Pitzschke et al., 2009) (see Recipes)

    Table 1. Oligonucleotides for RDSA
    Oligonucleotides for RDSA
    RDSA_1
    TAGTTGAGTCTCACAAACGAACAC(N20)CATTCCAAAATCCATGGCTGATA
    RDSA_1 fo
    TAGTTGAGTCTCACAAACGAACAC
    RDSA_1 re
    TATCAGCCATGGATTTTGGAATG
    RDSA_2
    AATGGATCCAAGCTTAAGC(N18)CGTTGAATTCCCATGGACA
    RDSA_2 fo
    AATGGATCCAAGCTTAAGC
    RDSA_2 re
    TGTCCATGGGAATTCAACG

Equipment

  1. Glutathione sepharose beads (GE Healthcare)
  2. The empty chromatography columns (Bio-Rad Laboratories)
  3. 4 °C refrigerator
  4. Centrifuge
  5. Whirling shaker

Procedure

  1. Random DNA preparation
    1. PCR amplification.
      The double strand DNA fragments RDSA1 and RDSA2 with 20- and 18-bp random sequence regions flanked by independent sets of adaptors were amplified on the templates of RDSA_1 and RDSA_2 by PCR, using RDSA_1/2fo and RDSA_1/2re primers, respectively (Table 1). Conditions for PCR amplification are: 5 min at 94 °C; 30 cycles of 30 sec at 94 °C, 30 sec at 50 °C, and 30 sec at 72 °C; 10 min at 72 °C.
    2. Purification of PCR products.
      1. Take at least 200 μl PCR products; add equal volume of 24:1 (chloroform: isoamyl alcohol) to sample tubes; shake tubes vigorously by hand for at least 30 sec, centrifuge the sample at 15,000 x g for 10 min at 4 °C.
      2. Transfer the aqueous phase to fresh tubes; add equal volume of isopropanol and mix; place in -20 °C freezer for 1 h.
      3. Centrifuge the samples at 15,000 x g for 10 min at 4 °C.
      4. Discard the supernatants and wash the precipitates with 70% alcohol.
      5. Air dry the precipitates for at least 10 min; dissolve the precipitates in sterile ddH2O and store at -20 °C until use.

  2. Prokaryotic expression of target protein tagged with GST or CBD (chitin binding domain)
    1. Construct expression vector.
      The coding region of the target gene is ligated in frame into the expression vector pGEX-6P-1 (for GST fusion protein) or pTXB3 (for CBD fusion protein).
    2. The constructs are transformed into E.coli expression strain BL21 (DE3) to express fusion proteins.

  3. RDSA cycles
    1. Harvest cells (100 ml bacterial cultures) and resuspend them in PBS buffer (for GST fusion protein) or column buffer (for CBD fusion protein).
    2. Break cells by sonication (950 W x 20%, 2-10 min according to the solubility of the expressed protein and with 2 sec interruption after every 2 sec sonication. The tube with sample should be placed in ice-water during manipulation) and centrifuge at 1,000 x g for 10 min at 4 °C.
    3. Prepare beads columns (load at least 3 ml beads in each chromatography column); equilibrate the glutathione sepharose beads column (for GST fusion protein) with PBS buffer (ten times of the bead volume), and the chitin beads column (for CBD fusion protein) with column buffer (ten times of the bead volume). In the following steps, the beads columns should be placed on ice.
    4. Slowly load the clarified lysate on to the columns.
    5. Wash the columns with at least 20 times of bed volume PBS buffer for GST fusion protein or column buffer for CBD fusion protein to thoroughly remove the unbound proteins; wash the beads at least three times with RDSA buffer; resuspend the beads in 3 ml RDSA buffer. Now the recombinant target protein is immobilized on beads.
    6. Divide the beads into 5-10 tubes (1.5 ml tube).
    7. Add the random DNA fragment RDSA1 or RDSA2 (10 μl, about 5-10 μg) into one tube; incubate for more than 2 h at 4 °C with gentle rolling.
    8. Centrifuge at 500 x g for 5 min at 4 °C, discard the supernatant completely.
    9. Wash the beads with RDSA buffer for at least four times to remove unbound DNA fragments; add 500 µl sterile ddH2O and boil the beads for 5 min to release the bound DNA; centrifuge at 500 x g for 5 min at 4 °C.
    10. Transfer the supernatant to a fresh tube; purify and precipitate the released DNA as described in step A2 (do not need to dissolve the DNA).
    11. Add PCR reagents to the tube:
      74 µl
      ddH2O
      10 µl
      10x PCR buffer
      10 µl
      2.5 mM dNTP
      2.5 µl
      20 μM RDSA1 fo or RDSA2 fo primer
      2.5 µl
      20 μM RDSA1 re or RDSA2 re primer
      1.0 µl
      rTaq DNA polymerase
      Perform PCR amplification using the following parameters: 5 min at 94 °C; 30 cycles of 30 sec at 94 °C, 30 sec at 50 °C, and 30 sec at 72 °C; 10 min at 72 °C.
    12. Purify the PCR products as described in A2; dissolve the DNA in 10 μl sterile ddH2O and transfer the DNA to another tube in C6 for the next RDSA cycle (repeat steps C7-12).
    13. After 5 to 10 cycles, clone the purified PCR products into pGEM-T easy vector and sequence.

Notes

  1. By increasing the amounts of the non-specific competitor poly (dIdC) (0, 50, 100, 150, 200, 250, 300 and 350 ng) and reducing the amounts of protein in the binding reactions (700 μl, 650 μl, 600 μl, 550 μl, 500 μl, 400 μl, 300 μl, 200 μl protein bound beads) the stringency can be increased in each RDSA cycle (Pitzschke et al., 2009; Wang et al., 2014).
  2. To exclude effects from the nonrandom flanking adaptors, the RDSAs must be performed in duplicates with two different sets of input random DNA fragments (RDSA1 and RDSA2) (Pitzschke et al., 2009).
  3. The number of total cycles may be different for different proteins. To our experience, at least 5 cycles should be performed to exclude the non-specific binding.
  4. The results of RDSAs should be verified by other methods, such as electrophoretic mobility shift assay (EMSA) and yeast one-hybrid system.
  5. The annealing temperature should be adjusted by different random primers.

Recipes

  1. PBS buffer (pH 7.3)
    140 mM NaCl
    2.7 mM KCl
    10 mM Na2HPO4
    1.8 mM KH2PO4
    5 mM DTT
    1 mM PMSF
  2. Column buffer
    20 mM Tris-HCl (pH 8.5)
    500 mM NaCl
    1 mM EDTA
  3. RDSA buffer
    5 mM Tris (pH 8.0)
    75 mM NaCl
    2.5 mM MgCl2
    0.5 mM EDTA
    5% glycerol
    1% Tween
    1 mM DTT

Acknowledgments

We thank Dr. Heribert Hirt for suggestions on this experiment. This work was supported by the Natural Science Foundation of China (grant no. 30971748).

References

  1. Pitzschke, A., Djamei, A., Teige, M. and Hirt, H. (2009). VIP1 response elements mediate mitogen-activated protein kinase 3-induced stress gene expression. Proc Natl Acad Sci U S A 106(43): 18414-18419.
  2. Wang, Y., Peng, W., Zhou, X., Huang, F., Shao, L. and Luo, M. (2014). The putative Agrobacterium transcriptional activator-like virulence protein VirD5 may target T-complex to prevent the degradation of coat proteins in the plant cell nucleus. New Phytol 203(4): 1266-1281.

简介

蛋白质-DNA相互作用是非常重要的细胞过程,通过其调节DNA生物学功能,通常是基因表达。 随机DNA结合选择测定(RDSA)的方法可以用于鉴定由具有DNA结合活性的蛋白质结合的DNA元件。 该方法基于固定的重组蛋白在特异性珠上的靶DNA元件的富集和重复PCR扩增。

关键字:蛋白质- DNA的相互作用, 转录因子, 顺式元件, 跨元素, DNA结合活性

材料和试剂

  1. 原核表达载体:pGEX-6P-1(GE Healthcare)或pTXB3(New England Biolabs)
  2. 表达菌株[大肠杆菌(
  3. T载体pGEMT-easy和T4-DNA连接酶(Promega Corporation,目录号:A1360)
  4. DNA聚合酶(Takara Bio公司,目录号:DR001)
  5. 无菌ddH 2 O(双蒸水)
  6. 寡核苷酸(Pitzschke等人,2009; Wang等人,2014)
  7. 壳多糖树脂(New England Biolabs,目录号:S6651S)
  8. PBS缓冲液(见配方)
  9. 列缓冲区(参见配方)
  10. RDSA缓冲区(Pitzschke等人,,2009)(参见Recipes)

    表1. RDSA 的寡核苷酸
    用于RDSA的寡核苷酸
    RDSA_1
    TAGTTGAGTCTCACAAACGAACAC(N20)CATTCCAAAATCCATGGCTGATA
    RDSA_1 fo
    TAGTTGAGTCTCACAAACGAACAC
    RDSA_1 re
    TATCAGCCATGGATTTTGGAATG
    RDSA_2
    AATGGATCCAAGCTTAAGC(N18)CGTTGAATTCCCATGGACA
    RDSA_2 fo
    AATGGATCCAAGCTTAAGC
    RDSA_2 re
    TGTCCATGGGAATTCAACG

设备

  1. 谷胱甘肽琼脂糖珠(GE Healthcare)
  2. 空色谱柱(Bio-Rad Laboratories)
  3. 4°C冰箱
  4. 离心机
  5. 旋转振动器

程序

  1. 随机DNA制备
    1. PCR扩增。
      双链DNA片段RDSA1和RDSA2 具有侧翼为独立集的20和18-bp随机序列区 通过PCR在RDSA_1和RDSA_2的模板上扩增适配子,   分别使用RDSA_1/2fo和RDSA_1/2re引物(表1)。 PCR扩增条件为:94℃5分钟; 30个循环的30 94℃,50℃30秒,72℃30秒; 72℃10分钟。
    2. PCR产物的纯化
      1. 取至少200μlPCR产物; 加入等体积的24:1 (氯仿:异戊醇)至样品管; 摇管大力   手持至少30秒,以15,000×g离心样品10分钟 min在4℃
      2. 将水相转移到新鲜管中; 加入等体积的异丙醇并混合; 置于-20°C冰箱1小时
      3. 在4℃下,将样品以15,000×g离心10分钟
      4. 弃去上清液,用70%酒精洗涤沉淀。
      5. 空气干燥沉淀至少10分钟; 将沉淀物溶解在无菌ddH 2 O中并在-20℃下储存直至使用。

  2. 用GST或CBD(壳多糖结合结构域)标记的靶蛋白的原核表达
    1. 构建表达载体。
      靶基因的编码区是   框内连接到表达载体pGEX-6P-1(用于GST融合) 蛋白质)或pTXB3(用于CBD融合蛋白)
    2. 将构建体转化到大肠杆菌表达菌株BL21(DE3)中以表达融合蛋白。

  3. RDSA周期
    1. 收获细胞(100ml细菌培养物),并将其重悬在PBS中 缓冲液(用于GST融合蛋白)或柱缓冲液(用于CBD融合) 蛋白质)。
    2. 通过超声破碎细胞(950W×20%,2-10分钟 根据表达蛋白的溶解度和2秒 每2秒超声处理后中断。 带样品的管应该 在操作期间置于冰水中)并在1,000℃下在4℃下离心10分钟。
    3. 准备珠柱(加载至少3毫升 珠); 平衡谷胱甘肽 Sepharose珠柱(用于GST融合蛋白) 倍的珠体积)和几丁质珠柱(用于CBD融合) 蛋白质)与柱缓冲液(珠体积的10倍)。 在里面 接下来的步骤中,珠子柱应该放在冰上
    4. 将澄清的裂解液缓慢加载到色谱柱上。
    5. 用至少20倍床体积PBS缓冲液洗涤柱子   GST融合蛋白或用于CBD融合蛋白的柱缓冲液 彻底去除未结合的蛋白; 洗珠至少三次 次; 将珠子重悬于3ml RDSA缓冲液中。 现在   重组靶蛋白固定在珠上。
    6. 将珠子分成5-10管(1.5 ml管)。
    7. 加入随机DNA片段RDSA1或RDSA2(10μl,约5-10μg) 成一管; 在4℃下温和滚动孵育2小时以上
    8. 在4℃下以500×g离心5分钟,完全丢弃上清液。
    9. 用RDSA缓冲液洗涤珠至少四次以除去 未结合的DNA片段; 加入500μl无菌ddH 2 O并煮沸珠5   min以释放结合的DNA; 在4℃下以500×g离心5分钟
    10. 将上清液转移到新管; 净化和沉淀 释放的DNA如步骤A2中所述(不需要溶解 脱氧核糖核酸)。
    11. 向试管中加入PCR试剂:
      74微升
      ddH sub 2 O
      10微升
      10x PCR缓冲液
      10微升
      2.5mM dNTP
      2.5μl
      20μMRDSA1 fo或RDSA2 fo primer
      2.5μl
      20μMRDSA1re或RDSA2引物
      1.0μl
      rTaq DNA聚合酶
      使用以下参数进行PCR扩增:94℃5分钟 C; 在94℃下30秒,在50℃下30秒和在72℃下30秒的30个循环; 72℃10分钟。
    12. 如A2中所述纯化PCR产物; 将DNA溶解在10μl无菌ddH 2 O中并将DNA转移至另一个 管在C6中用于下一个RDSA循环(重复步骤C7-12)。
    13. 5〜10个循环后,将纯化的PCR产物克隆到pGEM-T easy载体和序列中。

笔记

  1. 通过增加非特异性竞争剂聚(dIdC)(0,50,100,150,200,250,300和350ng)的量并减少结合反应中的蛋白质的量(700μl,650μl,600μl) 500μl,550μl,500μl,400μl,300μl,200μl蛋白结合珠),可以在每个RDSA循环中增加严格性(Pitzschke等人,2009; Wang等人 。,2014)。
  2. 为了排除来自非随机侧翼衔接子的影响,RDSA必须以两个不同组的输入随机DNA片段(RDSA1和RDSA2)重复进行(Pitzschke等人,2009)。
  3. 对于不同的蛋白质,总循环数可以不同。 根据我们的经验,应该进行至少5个循环以排除非特异性结合
  4. RDSAs的结果应该通过其他方法,如电泳迁移率变动分析(EMSA)和酵母单杂交系统验证。
  5. 退火温度应通过不同的随机引物调整

食谱

  1. PBS缓冲液(pH 7.3)
    140mM NaCl 2.7 mM KCl
    10mM Na 2 HPO 4
    1.8mM KH 2 PO 4 sub/
    5 mM DTT
    1mM PMSF
  2. 列缓冲区
    20mM Tris-HCl(pH8.5) 500 mM NaCl
    1mM EDTA
  3. RDSA缓冲区
    5mM Tris(pH8.0) 75mM NaCl 2.5mM MgCl 2 v/v 0.5mM EDTA 5%甘油 1%Tween
    1 mM DTT

致谢

我们感谢Heribert Hirt医生对本实验的建议。 这项工作是由中国自然科学基金会(拨款号30971748)支持。

参考文献

  1. Pitzschke,A.,Djamei,A.,Teige,M。和Hirt,H。(2009)。 VIP1反应元件介导促分裂原活化蛋白激酶3诱导的应激基因表达。 美国国家科学院院报106(43):18414-18419。
  2. Wang,Y.,Peng,W.,Zhou,X.,Huang,F.,Shao,L.and Luo,M.(2014)。 推定的土壤杆菌转录激活子样毒力蛋白VirD5可能靶向T复合物以防止外壳降解 蛋白质在植物细胞核中。新植物 203(4):1266-1281。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Wang, Y. and Luo, M. (2015). Random DNA Binding Selection Assay (RDSA). Bio-protocol 5(8): e1452. DOI: 10.21769/BioProtoc.1452.
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