RNA-protein interactions
RNA-protein interactions
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DOI：
10.29172/552ea8ca-3d9b-40a6-a22d-e2d6484c8880
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发表时间：
1994
期刊：
影响因子：
2.9
通讯作者：
L. B. Ludwig;Barbara J. Hughes;Stanley A. Schwartz
中科院分区：
文献类型：
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作者：
L. B. Ludwig;Barbara J. Hughes;Stanley A. Schwartz
凝胶阻滞或电泳迁移率变动分析（EMSA）是一种有用的方法，用于可视化DNA结合蛋白和DNA之间的特异性相互作用。DNA结合蛋白参与从转录和复制到重组和病毒整合的各种细胞过程。通常，在迁移率变动测定中使用含有与目的蛋白结合的序列的32 P标记的DNA探针，但先前已经描述了使用地高辛-dUTP标记的DNA的非放射性方法（1）。虽然生物素标记的探针的杂交已用于Southern或北方分析，但生物素标记的探针直接用于研究蛋白质-多核苷酸与EMSA的相互作用以前没有描述过。我们已经在这种非放射性EMSA中测试了一系列DNA大小，从63聚体Oct-I结合位点到含有HIV 1长末端重复序列（LTR）的690 bp dsDNA序列，以及生物素化的ssDNA和RNA探针（图1）。RNA、ssDNA或dsDNA探针的生物素化容易进行，并且所得探针稳定且灵敏，没有处理和处置放射性标记的寡核苷酸探针的危险。EMSA可以以标准方式进行，仅需要少量调整以检测生物素化探针，如下所述。合成的寡核苷酸是末端标记的或体外合成的，在如下合成反应期间将生物素1-UTP（Sigma Chemical Company，圣刘易斯Louis，MO）掺入RNA中或将生物素-16-dUTP（Boehringer-Mannheim Corporation，Indianapolis，IN）掺入ssDNA或dsDNA中。EBNA-1和Oct-I DNA是分别含有EB病毒核抗原（EBNA-1）和Oct-I蛋白的结合位点的82聚体和63聚体序列，得自Pharmacia LKB Biotechnology Inc.，皮斯卡特维，新泽西州（带移位套件）。白细胞介素-2（IL-2）增强子ClaI、HindIII片段从载体15 cxCAT中分离纯化，载体15 cxCAT是Gerald Crabtree博士赠送的礼物（2）。将含有5 ′突出端的DNA各自与40 μ M生物素-16-dUTP，dATP、dCTP和dGTP各20 μ M，在10 mMTris-HCl pH7.5、10 mMMgCl 2、50 mMNaCl、5 mMf-巯基乙醇和5 U DNA聚合酶I的Klenow片段中，在总体积50 μ l中，于37 ℃孵育1小时。HIV-1 dsDNA PCR产物由源自含有HIV-1长末端重复序列（LTR）和引物结合位点（PBS）的质粒的模板（pNLgag，来自NCI的Barbara Felber博士的馈赠）如下产生：使用限制酶从pNLgag切下包括LTR和PBS的DNA，然后从凝胶上洗脱。纯化的LTR（5 ′ U3-R-U 5-PBS 3 ′）用作聚合酶链式反应（PCR）的模板，沿着与LTR的U3、R或PBS区的+或链互补的合成引物。这些引物还含有T7或Sp 6 RNA聚合酶位点ssDNA。这些独特的引物用于产生和扩增dsDNA PCR产物，其还将在任一末端上掺入噬菌体DNA依赖性RNA聚合酶的位点。因为dsDNA PCR产物[LTR A（239 bp）和LTR B（690 bp）]在任一末端掺入DNA依赖性RNA聚合酶位点，所以我们可以将它们用作模板以任一方向合成RNA。T7或Sp 6 RNA聚合酶在RNA合成反应中与LTR A或B模板一起使用以产生相应的标记或未标记的RNA。然后，非生物素化的LTR A或LTR B衍生的RNA可以用作模板以产生相应的互补的生物素化的ssDNA HIV构建体。为了制备ssDNA，使用未标记的核糖核苷酸在体外合成RNA，然后用DNase处理去除最初的PCR产生的dsDNA模板，然后用苯酚-氯仿提取和乙醇沉淀。纯化的未标记的RNA模板然后用于在体外反应中合成标记的互补ssDNA，所述体外反应含有RNA模板、生物素-16-dUTP（Boehringer曼海姆）和dNTP，沿着相应的互补DNA寡核苷酸引物和Moloney鼠逆转录酶（Stratagene，拉霍亚，CA），如所述（3）。然后通过RNase H和RNase A处理从生物素化的ssDNA中去除RNA模板。将所有生物素化的dsDNA、ssDNA或RNA在水中平衡的Sephadex G-50柱上旋转，以分别除去未掺入的生物素-dUTP或未掺入的生物素-UTP。与含有来自EBNA-1蛋白的克隆DNA结合结构域的EBNA-1提取物的结合反应（Pharmacia BandShift试剂盒）或如（4）所述制备的Jurkat T细胞核提取物和~ 6 ng生物素化探针在20 mM Tris-HCl pH 7.5、100 mM NaCl、1 mM DTT、10%甘油、0.05%NP-40和50 ng聚（dI-dC）聚（dI-dC）在22 ℃下在总体积为20 μ l的溶液中反应20 min。在结合反应后，将10-20 μ l的结合混合物在5%丙烯酰胺凝胶上在lx TBE缓冲液中进行电泳，如（3）所述。在脱嘌呤后，
Gel retardation or electrophoretic mobility shift assay (EMSA) is a useful method for visualizing specific interactions between DNA-binding proteins and DNA. DNA-binding proteins are involved in a variety of cellular processes ranging from transcription and replication to recombination and viral integration. Typically, 32P-labeled DNA probes containing the sequence bound by the protein of interest are used in mobility shift assays, but a non-radioactive method using DNA labeled with digoxygenin-dUTP has previously been described (1). While hybridization of biotinylated probes has been employed in Southern or Northern analysis, use of biotin-labeled probes directly for studying protein-polynucleotide interactions with EMSA has not previously been described. We have tested a range ofDNA sizes, from a 63mer Oct-I binding site to a 690 bp dsDNA sequence containing the HIV1 long terminal repeat (LTR), as well as biotinylated ssDNA and RNA probes, in this non-radioactive EMSA (Fig. 1). Biotinylation ofRNA, ssDNA or dsDNA probes is easily performed, and the resultant probe is stable and sensitive, with none of the hazards of handling and disposing of radioactively-labeled oligonucleotide probes. EMSA can be performed in a standard fashion, with only a few adaptations required for detection ofthe biotinylated probe, as described below. Synthetic oligonucleotides were either end-labeled or synthesized in vitro, with incorporation of biotin1 -UTP (Sigma Chemical Company, St Louis, MO) into RNA or biotin-16-dUTP (Boehringer-Mannheim Corporation, Indianapolis, IN) into ssDNA or dsDNA during the synthesis reaction as follows. EBNA-1 and Oct-I DNAs are 82mer and 63mer sequences containing the binding sites for Epstein-Barr virus nuclear antigen (EBNA-1) and Oct-I protein, respectively, obtained from Pharmacia LKB Biotechnology Inc., Piscataway, NJ (Band Shift Kit). The interleukin-2 (IL-2) enhancer ClaI, HindlIl fragment was isolated and purified from the vector 15cxCAT, a kind gift from Dr Gerald Crabtree (2). DNAs containing 5' overhangs were each incubated with 40 ,uM biotin-16-dUTP, 20 jM each dATP, dCTP and dGTP, in 10mM Tris-HCl pH 7.5, 10 mM MgCl2, 50 mM NaCl, 5 mM f-mercaptoethanol and 5 U of the Klenow fragment ofDNA polymerase I and in a total volume of 50 ,ul at 37°C for I h. HIV-1 dsDNA PCR products were generated from a template derived from a plasmid containing the HIV-1 long terminal repeat (LTR) and the primer binding site (PBS), (pNLgag, a kind gift from Dr Barbara Felber, NCI) as follows: the DNA including LTR and PBS was cut out from pNLgag using restriction enzymes and then eluted from the gel. The purified LTR (5' U3-R-U5-PBS 3') was used as a template in polymerase chain reactions (PCR), along with synthesized primers complementary to + or strands of the U3, R or PBS regions of the LTR. These primers also contained either a T7 or Sp6 RNA polymerase site ssDNA. These unique primers were used to generate and amplify dsDNA PCR products that would also incorporate sites for bacteriophage DNA-dependent RNA polymerases on either end. Because the dsDNA PCR products [LTR A (239 bp) and LTR B (690 bp)] incorporated DNA-dependent RNA polymerase sites on either end, we could use them as templates to synthesize RNA in either orientation. T7 or Sp6 RNA polymerase were used with LTR A or B template in RNA synthesis reactions to generate the corresponding labeled or unlabeled RNA. The non-biotinylated LTR A or LTR B-derived RNA could then be used as template(s) to generate the corresponding, complementary, biotinylated, ssDNA HIV constructs. To make ssDNA, RNA was synthesized in vitro using unlabeled ribonucleotides, and then the original PCR-generated dsDNA template was removed with DNase treatment, followed by phenol-chloroform extraction and ethanol precipitation. The purified non-labeled RNA templates were then used to synthesize the labeled, complementary ssDNA in an in vitro reaction containing RNA template, biotin-16-dUTP (Boehringer Mannheim) and dNTPs, along with the corresponding, complementary DNA oligonucleotide primer and Moloney murine reverse transcriptase (Stratagene, La Jolla, CA) as described (3). TheRNA template could then be removed from the biotinylated ssDNA by RNase H and RNase A treatment. All biotinylated dsDNA, ssDNA orRNA were spun over a Sephadex G-50 column equilibrated in water to remove unincorporated biotin-dUTP or unincorporated biotin-UTP, respectively. Binding reactions with EBNA-1 extract containing the cloned DNA-binding domain from EBNA-1 protein (Pharmacia BandShift Kit) or Jurkat T cell nuclear extracts prepared as described (4) and -6 ng biotinylated probe were incubated in 20 mM Tris-HCl pH 7.5, 100 mM NaCl, 1 mM DTT, 10% glycerol, 0.05% NP-40 and 50 ng poly(dI.dC) poly(dI-dC) in a total volume of 20 gl for 20 min at 22°C. After the binding reaction, 10-20 p1 of the binding mixture was electrophoresed on a 5% acrylamide gel in lx TBE buffer as described (3). Following depurination,