Identification of RNA-binding Proteins by RNA Ligand-based cDNA Expression Library Screening

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Nucleic Acids Research
Jun 2014



We previously reported when a portion of the Requiem (REQ/DPF2) messenger ribonucleic acid (mRNA) 3’ untranslated region (3’UTR), referred to as G8, was overexpressed in K562 cells, β-globin expression was induced, suggesting that the 3’UTR of REQ mRNA plays a physiological role (Kim et al., 2014). To identify trans-acting factors that bind to the REQ 3’UTR, we describe the RNA ligand based cDNA expression library screening method. This protocol could be adapted to detect specific RNA-protein interactions. Following this method, we identified six positive clones in the initial round of screening and four pure clones after sib-screening. This protocol was originally published in Kim et al. (2014).

Keywords: RNA-protein interaction (RNA和蛋白质的相互作用), Phage display (噬菌体展示), Requiem (REQ/DPF2) (安魂曲(请求/ dpf2)), CDNA library screening (cDNA文库筛选)

Materials and Reagents

  1. X-Omat AR film (Eastman Kodak Company, catalog number: 0572842 )
  2. Eppendorf tubes
  3. K562 cells
    Note: Phagemid-based K562 cDNA expression libraries were constructed by isolating mRNA from cells with an Ultraspec-RNA isolating system and a biotinylated oligo (dT) probe.
  4. XL1-Blue cells (Agilent Technologies, catalog number: 200403 )
  5. XLOR cell (Agilent Technologies, catalog number: 200403 )
  6. 10% bovine calf serum (GE Healthcare, HycloneTM, catalog number: SH30073.03 )
  7. RPMI 1640 medium (Life Technologies, GibcoTM, catalog number: 11875-093 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 11875-093”.
  8. Ultraspec-RNA isolating system (Biotech, catalog number: BL-10050 )
  9. Biotinylated oligo (dT) probe (50 pmol/μl) (Promega Corporation, catalog number: Z5261 )
  10. cDNA synthesis kit (Agilent Technologies, catalog number: 200403 )
  11. λZAP II express phage vector (Agilent Technologies, catalog number: 200403 )
  12. Nitrocellulose membrane (immobilon-NC membrane) (EMD Millipore Corporation, catalog number: N8395 )
  13. Ribonucleic acid from torula yeast (RNA type VI) (Sigma-Aldrich, catalog number: R6625 )
  14. [α-32P]-labeled G8-RNA ligand (BMS)
  15. ExAssist helper phage (Agilent Technologies, catalog number: 200253 )
  16. IPTG [≥ 99% (TLC), ≤ 0.1% Dioxan] (Sigma-Aldrich, catalog number: I6758 )
  17. HEPES (pH 7.9)
  18. KCl
  19. 0.1% (w/v) Ficoll 400-DL
  20. 0.01% polyvinyl-pyrolidon PVP-40
  21. MnCl2
  22. ZnCl2
  23. EDTA
  24. DTT
  25. Tryptone
  26. NaCl
  27. Yeast Extract
  28. DW
  29. MgSO4
  30. Tris-HCl (pH 7.5)
  31. Gelatin
  32. Screening buffer (see Recipes)
  33. LB plates (see Recipes)
  34. SM buffer (see Recipes)


  1. Refrigerated Eppendorf centrifuge (Hanil, catalog number: Union 55R )
  2. Heat block (Thermolyne, catalog number: DB17615 )
  3. Tissue culture CO2 incubators set at 37 °C (HERAcell, catalog number: HERAcell® 240 )
  4. Developer (TAEAHN, catalog number: TM-90S )
  5. Vortex (JEIO TECH, catalog number: VM-96B )
  6. Deep freezer (Forma Scientific, catalog number: 917 )
  7. Shaker (SLB, catalog number: SLRM-3 )


  1. mRNA preparation
    Phagemid-based K562 cDNA expression libraries were constructed by isolating mRNA from cells with an Ultraspec-RNA isolating system and a biotinylated oligo (dT) probe. The detailed procedure is described below:
    1. Spin down K562 cells (5 ~ 10 x 106) at 5,000 rpm for 5 min at 4 °C.
    2. Add 1 ml of UltraspecTM RNA reagent to lyse cells by repetitive pipetting. Incubate at 4 °C for 5 min.
    3. Add 0.2 ml of chloroform and cover the samples tightly, shake vigorously for 15 sec and place on ice at 4 °C for 5 min.
    4. Centrifuge at 12,000 x g for 15 min at 4 °C.
    5. Carefully transfer the aqueous phase (about 4/5th volume) to a new fresh tube.
    6. Add equal volume of isopropanol and store sample for 10 min at 4 °C. Centrifuge at 12,000 x g for 10 min at 4 °C.
    7. Remove the supernatant and wash RNA pellet twice with 1 ml of 75% ethanol by vortexing and subsequent centrifuge for 5 min at 7,500 x g at 4 °C.
    8. Remove the supernatant and briefly air dry the RNA pellet for 5 min. Dissolve the RNA pellet in 50-100 ul of Ultraspec DEPC treated water by vortexing for 1 min. Measure RNA concentration using Nanodrop.

  2. cDNA library construction
    Double stranded cDNA was synthesized using a cDNA synthesis kit with an oligo(dT) linker primer, and the resultant cDNA was ligated into the λZAP II express phage vector. The detailed procedure is described below:
    1. Set up a positive control ligation to ligate the test insert into the ZAP Express vector as follows:
      1. 1 μl of the ZAP Express vector (1 μg)
      2. 1.6 μl of test insert (0.4 μg)
      3. 0.5 μl of 10x ligase buffer
      4. 0.5 μl of 10 mM rATP (pH 7.5)
      5. 0.9 μl of water
      6. Then add 0.5 μl of T4 DNA ligase (4 U/μl)
    2. To prepare the sample ligation, add the following components:
      1. 2 μl of resuspended cDNA (~100 ng)
      2. 0.5 μl of 10x ligase buffer
      3. 0.5 μl of 10 mM rATP (pH 7.5)
      4. 1.0 μl of the ZAP Express vector (1 μg/μl)
      5. 0.5 μl of water for a final volume of 4.5 μl
      6. Then add 0.5 μl of T4 DNA ligase (4 U/μl)
    3. Incubate the reaction tubes overnight at 4 °C.
    4. After ligation is complete, package 1 μl of each ligation using Gigapack III Gold packaging extract in the λZAP II express phage vector package to produce transduced lambda phages according to the packaging instructions outlined in Packaging.

  3. Library plating
    1. Add 1 μl of the lambda phage to 200 μl of XL1-Blue host cells.
    2. Incubate the phage and bacteria for 15 min at 37 °C to allow the phage to attach to the cells.
    3. Phagemids (1-1.5 x 104 pfu/plate) were inoculated onto LB plates harboring a lawn of XL1-Blue cells for 4 h incubation at 37 °C.

  4. Plaque lifts
    1. Carefully place the nitrocellulose filter (contain 20 mM IPTG) on top of the plate (not letting any bubbles to form between the nitrocellulose and the plate.
    2. Leave filter for 10 h on the plate.

  5. Prehybridization and Hybridization reactions
    1. The membranes were blocked in screening buffer containing 0.1 mg/ml yeast RNA in order to reduce nonspecific binding of the probe RNA.
    2. Specific RNA-protein interactions were detected by hybridization with the [α-32P]-labeled G8-RNA ligand (~ 0.5 x 106 cpm/ml) in screening buffer.
    3. Let hybridization go 2 h at 4 °C.

  6. Washes
    1. Non-specific bound radioactivity was removed by washing four times at room temperature in 100 ml screening buffer for 5 min each. (Check the membranes for radioactivity using Mini 900 Scintillation monitor.) If it is low (less than 1 count per second), stop here; otherwise, if the signal is still too high (higher than 1 count per second), do the last, stringent, wash).
    2. Remove membranes and let dry for > 10 min.

  7. Exposures
    1. Cover membranes with plastic wrap.
    2. In the dark, place Kodak X-Omat AR film on top of membranes and tape the film to prevent it from moving out of position.
    3. Leave at -70 °C for 24 h (don't use cassettes without intensifying screens).
    4. Develop film.

  8. Plaque isolation and Clone purification
    1. Using the developed films, align the plates with the developed film and isolate a positive plaque.
    2. Place the plug in 1 ml of SM buffer and store at 4 °C.
    3. Elute the phage from the plug by rocking O/N at 4 °C.
    4. Repeat screening rounds until a single plaque can be isolated that when spread on a plate and probed will show that all the plaques are positives (usually 3 screens are enough to achieve this).

  9. Excision of plasmids from phage
    Each positive phagemid was converted into a plasmid (pBK-CMV) by inoculating the phage into XLOR cells along with the ExAssist helper phage. The detailed procedure is described below:
    1. The plaque of interest from the agar plate and transfer the plaque to a sterile centrifuge tube containing 500 μl of SM buffer and 20 μl of chloroform.
    2. Vortex the centrifuge tube to release the phage particles into the SM buffer. Incubate the centrifuge tube for overnight at 4 °C.
    3. Grow separate 50 ml overnight cultures of XL1-Blue and SOLR cells in LB broth with supplements at 30 °C. Gently spin down the XL1-Blue and SOLR cells (1,000 x g). Resuspend each of the cell pellets in 25 ml of 10 mM MgSO4.
    4. Combine the following components in a 15 ml BD Falcon polypropylene round-bottom tube:
      1. 200 μl of XL1-Blue cells at an OD600 of 1.0
      2. 250 ul of phage stock (containing >1 x 105 phage particles)
      3. 1 μl of the ExAssist helper phage (>1 x 106 pfu/μl)
    5. Incubate at 37 °C for 15 min to allow the phage to attach to the cells.
    6. Add 3 ml of LB broth with supplements and incubate for 2.5-3 h at 37 °C with shaking.
    7. Heat the tube at 65-70 °C for 20 min to lyse the lambda phage particles and the cells. Spin the tube at 1,000 x g for 15 min to pellet the cell debris.
    8. Decant the supernatant into a sterile 15 ml tube. This stock contains the excised pBluescript phagemid packaged as filamentous phage particles.
    9. To plate the extracted phagemids, add 200 μl of freshly grown SOLR cells from step I3 to two 1.5 ml centrifuge tubes. Add 100 μl of the phage supernatant to one centrifuge tube and 10 μl of the phage supernatant to the other centrifuge tube.
    10. Incubate the centrifuge tubes at 37 °C for 15 min. Plate 200 μl of the cell mixture from each centrifuge tube on LB-ampicillin agar plates (100 μg/ml) and incubate the plates overnight at 37 °C.
    11. Positive clones were identified by sequencing.

Representative data

Figure 1. Scheme of RNA-Ligand based cDNA expression library screening

Figure 2. Autoradiograph showing positive putative clones after primary screening. All RNA-binding proteins were additionally confirmed by secondary/tertiary screening.


Figure 3. Autoradiography showing high background due to the low concentration of competitor yeast RNA (step E) and poor washing of non-specific bound radioactivity (step F)


  1. Screening buffer
    15 mM HEPES (pH 7.9)
    50 mM KCl
    0.1% (w/v) Ficoll 400-DL
    0.01% polyvinyl-pyrolidon PVP-40
    0.1 mM MnCl2
    0.1 mM ZnCl2
    0.1 mM EDTA
    0.5 mM DTT
  2. LB plate
    1.0 g Tryptone
    1.0 g NaCl
    0.5 g Yeast Extract
    DW 100 ml
  3. SM buffer
    5.8 g NaCl
    2 g MgSO4
    50 mM Tris HCl (pH 7.5)
    100 mg Gelatin
    DW 1 L


This work was supported by Basic Science Research Program (2010-00252250 to C. G. K.), National Research Foundation (NRF), Ministry of Education, Science and Technology (MEST), Republic of Korea. Converging Research Center Program (2013K000283 to C. G. K.), Ministry of Science, ICT & Future Planning (MSIFP), Republic of Korea.


  1. Sagesser, R., Martinez, E., Tsagris, M. and Tabler, M. (1997). Detection and isolation of RNA-binding proteins by RNA-ligand screening of a cDNA expression library. Nucleic Acids Res 25(19): 3816-3822.
  2. Sanger, F., Nicklen, S. and Coulson, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A 74(12): 5463-5467.


我们先前报道当在K562细胞中过表达Requiem(REQ/DPF2)信使核糖核酸(mRNA)3'非翻译区(3'UTR),称为G8的一部分时,诱导β-球蛋白表达, REQ mRNA的3'UTR起生理作用(Kim等人,2014)。 为了鉴定结合REQ 3'UTR的反式作用因子,我们描述了基于RNA配体的cDNA表达文库筛选方法。 这个协议可以适应于检测特定的RNA-蛋白相互作用。 按照这种方法,我们在初始轮筛选中鉴定了6个阳性克隆,在同胞筛选后鉴定了4个纯克隆。 该协议最初发表于Kim等人(2014)。

关键字:RNA和蛋白质的相互作用, 噬菌体展示, 安魂曲(请求/ dpf2), cDNA文库筛选


  1. X-Omat AR膜(Eastman Kodak Company,目录号:0572842)
  2. Eppendorf管
  3. K562细胞
    注意:通过用Ultraspec-RNA分离系统和生物素化的oligo(dT)探针从细胞中分离mRNA来构建基于噬菌粒的K562 cDNA表达文库。
  4. XL1-Blue细胞(Agilent Technologies,目录号:200403)
  5. XLOR细胞(Agilent Technologies,目录号:200403)
  6. 10%牛小牛血清(GE Healthcare,Hyclone TM,目录号:SH30073.03)
  7. RPMI 1640培养基(Life Technologies,Gibco TM ,目录号:11875-093)
    注意:目前,"Thermo Fisher Scientific,Gibco TM
  8. Ultraspec-RNA分离系统(Biotech,目录号:BL-10050)
  9. 生物素化的寡(dT)探针(50pmol/μl)(Promega公司,目录号:Z5261)
  10. cDNA合成试剂盒(Agilent Technologies,目录号:200403)
  11. λZAPII表达噬菌体载体(Agilent Technologies,目录号:200403)
  12. 硝化纤维素膜(immobilon-NC膜)(EMD Millipore公司,目录号:N8395)
  13. 来自torula酵母的核糖核酸(RNA VI型)(Sigma-Aldrich,目录号:R6625)
  14. [α-32P] - 标记的G8-RNA配体(BMS)
  15. ExAssist辅助噬菌体(Agilent Technologies,目录号:200253)
  16. IPTG [≥99%(TLC),≤0.1%二恶烷](Sigma-Aldrich,目录号:I6758)
  17. HEPES(pH 7.9)
  18. KCl
  19. 0.1%(w/v)Ficoll 400-DL
  20. 0.01%聚乙烯吡咯烷酮PVP-40
  21. MnCl 2
  22. ZnCl 2
  23. EDTA
  24. DTT
  25. 胰蛋白酶
  26. NaCl
  27. 酵母提取物
  28. DW
  29. MgSO 4 4 /
  30. Tris-HCl(pH 7.5)
  31. 明胶
  32. 筛选缓冲区(请参阅配方)
  33. LB板(见配方)
  34. SM缓冲区(参见配方)


  1. 冷冻Eppendorf离心机(Hanil,目录号:Union 55R)
  2. 加热块(Thermolyne,目录号:DB17615)
  3. 设定在37℃的组织培养CO 2培养箱(HERAcell,目录号:HERAcell 240)
  4. 显影剂(TAEAHN,目录号:TM-90S)
  5. Vortex(JEIO TECH,目录号:VM-96B)
  6. 深冷冻箱(Forma Scientific,目录号:917)
  7. Shaker(SLB,目录号:SLRM-3)


  1. mRNA制备
    通过用Ultraspec-RNA分离系统和生物素化的寡(dT)探针从细胞中分离mRNA构建基于噬菌粒的K562 cDNA表达文库。详细步骤如下:
    1. 在4℃下以5,000rpm旋转K562细胞(5?10×10 6次)达5分钟。
    2. 加入1毫升的Ultraspec TM RNA试剂通过重复移液裂解细胞。在4℃孵育5分钟。
    3. 加入0.2ml氯仿并盖紧样品,剧烈摇动15秒,置于冰上4℃5分钟。
    4. 在4℃下以12,000xg离心15分钟。
    5. 小心地将水相(约4/5体积)转移到新的新管中
    6. 加入等体积的异丙醇,并在4℃下存储样品10分钟。在4℃下以12,000xg离心10分钟。
    7. 除去上清液,洗涤RNA沉淀两次,用1ml的75% 乙醇,通过涡??旋和随后在7,500×g /孔离心5分钟 ?4℃。
    8. 除去上清液,短暂空气干燥RNA沉淀 5分钟。将RNA沉淀溶解在50-100ul的Ultraspec DEPC中 处理的水通过涡旋1分钟。使用测量RNA浓度 Nanodrop。

  2. cDNA文库构建
    1. 建立阳性对照连接以将测试插入片段连接到ZAP Express载体中,如下:
      1. 1μlZAP Express载体(1μg)
      2. 1.6μl测试插入物(0.4μg)
      3. 0.5μl10x连接酶缓冲液
      4. 0.5μl10mM rATP(pH7.5)
      5. 0.9μl水
      6. 然后加入0.5μlT4 DNA连接酶(4 U /μl)
    2. 要准备样品连接,请添加以下组件:
      1. 2μl重悬的cDNA(?100ng)
      2. 0.5μl10x连接酶缓冲液
      3. 0.5μl10mM rATP(pH7.5)
      4. 1.0μlZAP Express载体(1μg/μl)
      5. 0.5μl水,最终体积为4.5μl
      6. 然后加入0.5μlT4 DNA连接酶(4 U /μl)
    3. 孵育反应管在4℃过夜。
    4. 连接完成后,包装1μl每个连接使用 Gigapack III金包装提取物在λZAPII表达噬菌体载体 包装根据包装产生转导的λ噬菌体 包装中概述的说明。

  3. 图书馆电镀
    1. 加入1μlλ噬菌体至200μlXL1-Blue宿主细胞
    2. 孵育噬菌体和细菌15分钟,在37℃,以允许噬菌体附着到细胞。
    3. 将噬菌粒(1-1.5×10 4 pfu /平板)接种到LB平板上 包含XL1-Blue细胞的草坪,在37℃温育4小时。

  4. 斑块升高
    1. 小心地将硝酸纤维素滤膜(含20 mM IPTG)放在上面 ?的板(不让任何气泡之间形成 硝化纤维素和板。
    2. 在板上留下过滤器10小时。

  5. 预杂交和杂交反应
    1. 将膜在含有0.1mg/ml的筛选缓冲液中封闭 酵母RNA,以减少探针RNA的非特异性结合。
    2. 通过与杂交检测特异性RNA-蛋白相互作用 ?在筛选中[α-32 P] - 标记的G8-RNA配体(?0.5×10 6 cfu/ml) 缓冲区
    3. 让杂交在4℃下2小时。

  6. 洗涤
    1. 通过在4℃洗涤4次除去非特异性结合放射性 ?室温在100ml筛选缓冲液中每次5分钟。 (检查 使用Mini 900闪烁监测器的放射性膜。如果它 低(每秒少于1个计数),停在这里;否则,如果 信号仍然太高(高于每秒1次计数),做最后一次, 严格,洗涤)。
    2. 取出膜,干燥> 10分钟。

  7. 曝光
    1. 用塑料包膜覆盖膜。
    2. 在黑暗中,将Kodak X-Omat AR膜放在膜的顶部,并胶带以防止其移出位置。
    3. 在-70°C保持24小时(不要使用没有增强屏幕的盒式磁带)。
    4. 发展电影。

  8. 斑块分离和克隆纯化
    1. 使用显影的胶片,将显影片与显影胶片对齐,并分离阳性斑块
    2. 将插头放入1 ml SM缓冲液中,在4°C下保存
    3. 通过在4°C摇动O/N,从噬菌体中洗脱噬菌体。
    4. 重复筛选轮,直到可以分离单个斑块 当在板上展开并探测将显示所有的斑块 积极(通常3个屏幕就足以实现这一点)。

  9. 从噬菌体中切除质粒
    1. 从琼脂平板感兴趣的斑块并将斑块转移到 ?含有500μlSM缓冲液和20μl的无菌离心管 氯仿。
    2. 涡旋离心管以释放噬菌体 颗粒进入SM缓冲液。孵育离心管过夜 ?在4℃。
    3. 生长XL1-Blue的单独50ml过夜培养物 ?SOLR细胞在LB肉汤中的补充物在30℃。轻轻向下旋转 XL1-Blue和SOLR细胞(1,000×g )。重悬每个细胞沉淀 在25ml的10mM MgSO 4中的溶液中。
    4. 将以下组分在15ml BD Falcon聚丙烯圆底管中混合:
      1. 将200μlXL1-Blue细胞在OD 600 600为
      2. 250ul噬菌体原液(含有> 1×10 5个噬菌体颗粒)
      3. 将1μl的ExAssist辅助噬菌体(> 1×10 6 pfu /μl)
    5. 在37℃孵育15分钟,以允许噬菌体附着到细胞。
    6. 加入3毫升带有补充物的LB肉汤,并在37℃下振荡孵育2.5-3小时。
    7. 在65-70℃加热管20分钟以裂解λ噬菌体 颗粒和细胞。在1000×g下旋转管15分钟以沉淀 ?细胞碎片
    8. 倾析上清液到无菌15毫升 管。该库存包含切下的pBluescript噬菌粒 丝状噬菌体颗粒。
    9. 为了铺板提取的噬菌粒, 加入200μl来自步骤I3的新鲜生长的SOLR细胞至两个1.5ml 离心管中。加入100微升的噬菌体上清液到一个离心机 管和10μl的噬菌体上清液到另一个离心管中。
    10. 孵育离心管在37℃15分钟。板200微升 将来自每个离心管的细胞混合物置于LB-氨苄青霉素琼脂平板上 (100μg/ml),并在37℃下将板温育过夜
    11. 通过测序鉴定阳性克隆。







  1. 筛选缓冲区
    15mM HEPES(pH 7.9)
    50 mM KCl
    0.1%(w/v)Ficoll 400-DL
    0.01%聚乙烯吡咯烷酮PVP-40 0.1mM MnCl 2/
    0.1mM ZnCl 2 2 / 0.1mM EDTA
    0.5 mM DTT
  2. LB平板
    1.0克NaCl 0.5 g酵母提取物
    DW 100 ml
  3. SM缓冲区
    5.8克NaCl 2g MgSO 4 50mM Tris HCl(pH7.5) 100mg明胶
    DW 1 L


这项工作得到大韩民国教育,科学和技术部(MEST)国家研究基金会(NRF)的基础科学研究计划(2010-00252250至C. G. K.)的支持。融合研究中心计划(2013K000283 to C. G. K.),Ministry of Science,ICT&未来规划(MSIFP),大韩民国。


  1. Sagesser,R.,Martinez,E.,Tsagris,M。和Tabler,M。(1997)。 通过RNA表达文库的RNA配体筛选检测和分离RNA结合蛋白。 a> Nucleic Acids Res 25(19):3816-3822
  2. Sanger,F.,Nicklen,S。和Coulson,A.R。(1977)。 DNA测序与链终止抑制剂。 Proc Natl Acad Sci USA 74(12):5463-5467。
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引用:Kim, M. Y., Lee, J. J. and Kim, C. G. (2016). Identification of RNA-binding Proteins by RNA Ligand-based cDNA Expression Library Screening. Bio-protocol 6(2): e1715. DOI: 10.21769/BioProtoc.1715.