Purification of RNA Mango Tagged Native RNA-protein Complexes from Cellular Extracts Using TO1-Desthiobiotin Fluorophore Ligand
使用TO1-脱硫生物素荧光团配体从细胞提取物中纯化RNA Mango标记的天然RNA-蛋白质复合物   

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RNA
Jul 2017

 

Abstract

A native purification strategy using RNA Mango for RNA based purification of RNA-protein complexes is described. The RNA Mango aptamer is first genetically engineered into the RNA of interest. RNA Mango containing complexes obtained from cleared cellular native extracts are then immobilized onto TO1-Desthiobiotin saturated streptavidin agarose beads. The beads are washed to remove non-specific complexes and then the RNA Mango containing complexes are eluted by the addition of free biotin to the beads. Since the eluted complexes are native and fluorescent, a second purification step such as size exclusion chromatography can easily be added and the purified complexes tracked by monitoring fluorescence. The high purity native complexes resulting from this two-step purification strategy can be then used for further biochemical characterization.

Keywords: RNA Mango (RNA Mango), TO1-Desthiobiotin (TO1脱硫生物素), RNP complex purification (RNP复合物纯化), Fluorophore (荧光团), Native purification (非变性纯化), Non-coding RNAs (非编码RNA), RNA-protein complexes (RNA-蛋白质复合物)

Background

Current RNA tags suffer from limitations such as poor KD, large size, potential biological interference or lack of intrinsic fluorescence (Panchapakesan et al., 2015). RNA Mango is small, can be simply integrated into stem-loop structures, in particular, GNRA tetraloops, is biologically tolerated and above all has a high affinity for its thiazole orange-based (TO1) ligand, TO1-Desthiobiotin (TO1-Dtb). This allows Mango tagged complexes to be easily bound and washed on streptavidin beads (summarized in Figure 1). The Mango:TO1-Desthiobiotin complex is highly fluorescent and can be eluted from streptavidin beads by the addition of biotin. The fluorescence of Mango tagged native complexes allows additional purification steps to be used to obtain highly purified native complexes.


Figure 1. Purification of Mango tagged RNA-protein complexes out of native extract. RNA constructs are designed (Procedure A, Figure 2) so that the Mango tag is located in a biologically compatible location. After bacterial expression of this construct, a native extract containing the Mango tagged RNA (Mango tag shown in red highlight), is prepared (Procedure B). Mango tagged RNA and RNA complexes are then bound to streptavidin beads (Black circle containing S) that have been derivatized with TO1-Desthiobiotin (Dtb, Procedure C). The thiazole orange moiety of TO1-Dbt is shown in purple and becomes highly fluorescent once bound by Mango (bright red highlight). Bound complexes are then extensively washed in native conditions (Procedure D). After washing fluorescent Mango tagged complexes can be eluted in native conditions by addition of biotin (Procedure E). Downstream analysis including further purification steps can then be simply implemented (Procedures F and G).

Materials and Reagents

  1. Low retention pipette tips (Fisher Scientific, catalog numbers: 02-717-134 , 02-717-143 , 02-707-511 )
  2. Microcentrifuge tubes, pre-lubricated (Corning, Costar®, catalog numbers: 3206 , 3207 )
  3. 96-well black wall, clear bottom, non-binding, non-sterile, Greiner Bio-One microtiter plate (Greiner Bio One International, catalog number: 655906 )
  4. Tricorn XK 16/40 SEC column (GE Healthcare, catalog number: 28988938 )
  5. Bacterial cells (E. coli) with RNA Mango tagged RNA of interest
  6. Liquid nitrogen
  7. TO1-PEG3-Desthiobiotin (ABM, catalog number: G956 )
  8. High capacity streptavidin agarose beads (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 20357 )
  9. Superdex 200 resin (GE Healthcare, catalog number: 17104301 )
  10. SYBR Green II RNA Gel Stain (Thermo Fisher Scientific, InvitrogenTM, catalog number: S7564 )
  11. 19:1 Acrylamide:Bis 40% (Thermo Fisher Scientific, catalog number: AM9022 )
  12. N,N,N’,N’-Tetramethylethylenediamine (TEMED) (Sigma-Aldrich, catalog number: T9281 )
  13. Ammonium persulphate (APS) (Bio-Rad Laboratories, catalog number: 1610700 )
  14. Boric acid (ACP Chemicals, catalog number: B2940 )
  15. EDTA (ACP Chemicals, catalog number: E4320 )
  16. Tris base (Fisher Scientific, catalog number: BP152 )
  17. Tris-HCl (Sigma-Aldrich, Roche Diagnostics, catalog number: 10812846001 )
  18. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
  19. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266 )
  20. DTT (Sigma-Aldrich, Roche Diagnostics, catalog number: 10197777001 )
  21. Sodium hydroxide (NaOH) (VWR, BDH, catalog number: BDH9292 )
  22. Sodium chloride (NaCl) (ACP Chemicals, catalog number: S2830 )
  23. HEPES (Sigma-Aldrich, catalog number: H3375 )
  24. Heparin (Sigma-Aldrich, catalog number: H0777-25KU )
  25. Biotin (Sigma-Aldrich, catalog number: B4501 )
  26. Yeast extract (Fisher Scientific, catalog number: BP1422 )
  27. Tryptone (Fisher Scientific, catalog number: BP1421 )
  28. Potassium hydroxide (KOH) (VWR, BDH, catalog number: BDH9262 )
  29. 10x Bacterial Native Extract (BNE) buffer (see Recipes)
  30. 10x Buffer A (see Recipes)
  31. 10x HEPES KCl (HK) buffer (see Recipes)
  32. 10x Binding buffer (see Recipes)
  33. 10x Biotin elution buffer (see Recipes)
  34. LB media (see Recipes)

Equipment

  1. Standard personal protective equipment
  2. Pipettes
  3. Peristaltic pump
  4. Fraction collector and appropriate collection tubes
  5. SpectraMax M5 fluorescent plate reader (Molecular Devices, model: SpectraMax M5 )
  6. Temperature controlled room (To be maintained at 4 °C)
  7. Preparative centrifuge, microcentrifuge and benchtop Picofuge
  8. Tube rotator for 1.5 ml tubes
  9. Polyacrylamide gel running equipment
  10. French press
  11. Sorvall RC6+ centrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: SorvallTM RC 6 Plus )
  12. Sorvall SLA-1500 rotor (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: SLA-1500 )
  13. NanoDrop
  14. Autoclave

Procedure

  1. Construct design
    1. A single copy of RNA Mango is usually sufficient for the pull down analysis and can be inserted into pre-existing stem loops that do not interfere with the function of the RNA of interest (Figure 2 (Panchapakesan et al., 2017; Trachman III et al., 2017). If inserting into a pre-existing stem loop structure, the RNA Mango quadruplex core and GAAA tetraloop-like isolation domain then the Mango aptamer can be inserted within the stem structure using the following sequence: 5’-...NNN NNN GAA GGG ACG GUG CGG AGA GGA GAN’ N’N’N’ N’N’.., where the left arm of the arbitrary RNA helix is indicated by N residues. The right arm (being the reverse complement of the left) is indicated by N’ symbols. The GAAA tetraloop-like isolation motif (shown in italics) together with the TO1-Desthiobiotin binding quadruplex core (in bold) being inserted within the arbitrary helix. While GNRA tetraloops are common in nature, RNA Mango can also be inserted into the 3’ or 5’ end of an unstructured RNA of interest. In this case, RNA Mango (including the GAAA tetraloop-like motif) together with an arbitrary stem should be inserted. New Mango tags (Mango II, III and IV) can be inserted in a similar fashion (Autour et al., 2018).


      Figure 2. Design of biologically compatible Mango-I tags. A. A general GNRA tetraloop structure is shown, depicted using Leontis-Westhof nomenclature (Leontis and Westhof, 2001). B. Structure of Mango I (figure modified from Autour et al. (2018)). Mango can be incorporated into naturally occurring stem-loop structures by replacing the N-N’ stem highlighted in purple with the stem sequence of the RNA of interest. In Mango I, a GAAA tetraloop-like motif (blue) encloses the core G-quadruplex (3 tiers: T1, T2, T3) by flanking it such that the Mango I core fits into GAA/A, where ‘/’ is the point of the Mango core insertion. Thus, Mango I can be inserted into a pre-existing GNRA tetraloop structure in a similar manner, so long as Mango does not interfere with the tetraloop’s biological function.

    2. The Mango tagged RNA should be tested for the functionalities of the Mango tag as well as the RNA of interest. The functionality of the Mango tag can be tested by measuring the fluorescence of the Mango tagged RNA of interest and comparing it to in vitro transcribed RNA Mango (Panchapakesan et al., 2017). Typically, a 1 µM solution of RNA is made along with two-fold excess of TO1-Dtb ligand in 1x Binding buffer. After incubating at RT for 5 min, fluorescence assays can be performed using a fluorometer. For a SpectraMax M5 fluorometer, the following excitation and emission setup is optimal for analyzing RNA Mango mediated fluorescence: Excitation at 495 nm; Emission 535 nm PMT: Medium, Cut-off 515 nm–Bottom read. Using a SpectraMax M5 under the aforementioned settings, RNA Mango concentrations as low as 5 nM can be reliably detected. The user should optimize the excitation and emission wavelengths to minimize interference from the excitation light source of the specific fluorometer being used. 

  2. Native extract preparation
    1. Bacterial cells (E. coli) with RNA Mango tagged RNA of interest should be grown in appropriate growth conditions to maximize the formation of the RNP complex desired. We typically grew cells overnight in 1 L LB media.
    2. Cells are then pelleted at 3,800 x g (RCF) for 10 min in a refrigerated Sorvall RC6+ centrifuge, using a Sorvall SLA-1500 rotor. The resulting cell pellet is then re-suspended in 20 ml of pre-chilled BNE buffer in ice. Cells are lysed by passaging through a 4 °C French press twice at 1,000 psi. If a French press is unavailable, then alternative cell disruption techniques such as sonication or enzymatic methods such as lysozyme digestion can be used. This crude lysate is then cleared by centrifugation at 13,200 x g (RCF) for 10 min at 4 °C. The cleared lysate is flash frozen in liquid nitrogen and stored at -80 °C until ready for use. For other organisms, including yeast, appropriate native extract preparation protocols should be used.
    3. Ensure the presence of KCl (90 to 150 mM), depending upon the stability of RNP complex of interest, as binding to TO1-Desthiobiotin requires KCl (Dolgosheina et al., 2014; Jeng et al., 2016; Panchapakesan et al., 2017). Handle all material in Steps B2 and B3 PROMPTLY on ice.

  3. TO1-Desthiobiotin streptavidin agarose bead preparation
    Prepare an appropriate amount of streptavidin agarose bead slurry. Using 7 ml of bacterial extract and 700 μl of streptavidin agarose bead slurry, derivatized with 42 nanomoles of TO1-Desthiobiotin, we obtained high quality results when overexpressing the 6S regulatory RNA (Dolgosheina et al., 2014; Panchapakesan et al., 2017). Scale the protocol below to suit experimental conditions.
    1. Carefully re-suspend streptavidin agarose bead slurry by gentle agitation.
    2. Take up 700 μl streptavidin agarose beads in a 1.5 ml Eppendorf tube.
    3. Wash twice at room temperature (RT) with 1 ml Buffer A, 2 min each, using a standard laboratory rotator. The tubes can be centrifuged briefly in a Picofuge to remove excess liquid from the beads.
    4. Wash beads three times using 1 ml HK buffer for 2 min each wash, again using a rotator.
    5. For every 100 μl streptavidin agarose beads incubate with 6 nanomoles of TO1-Desthiobiotin in 100 μl of HK buffer. Incubate at RT for 15 min on a rotator. Note the OD500500 = 63,000 M-1 cm-1) of the fluorophore solution before and after binding to the beads to estimate the amount of fluorophore bound using a NanoDrop. More than 90% of TO1-Desthiobiotin should be bound. The color of the beads will now turn orange because of the fluorophore binding. Different beads may have distinct binding capacities, which should be empirically determined if needed.
    6. Wash with 1 ml of HK buffer to remove residual unbound fluorophore.

  4. Binding of extract to prepared agarose beads
    Since the final goal of this procedure is to purify RNP complexes in native conditions, care should be taken not to denature the complex in every step of the purification viz., bead binding, washing and elution. Since RNA Mango requires KCl for proper folding and binding to the TO1-Desthiobiotin ligand (Dolgosheina et al., 2014; Jeng et al., 2016; Trachman III et al., 2017), KCl should be included in all binding, washing and elution steps. Other components of the buffer can be modified according to experimental needs.
    1. Bind 7 ml of bacterial extract to beads at RT. Incubate at RT for 15 min on a rotator (in the case of yeast extract (Panchapakesan et al., 2017), binding is performed at 4 °C, to ensure stabilization of the RNP complex). The extract can be diluted, if required, by the addition of BNE or Binding buffer to the extract.
    2. Wash twice with 1 ml Binding buffer for 2 min each on a rotator. The number of washes selected depends on the anticipated stability of the RNP complex. Likewise, the temperature (4-37 °C) and time (2-15 min) of each wash can be significantly adjusted if optimization is required.

  5. Biotin-mediated native elution
    Elute using 20 mM Biotin elution buffer in 500 μl volume at 37 °C (Panchapakesan et al., 2017) for 20 min on a rotator as biotin effectively displaces desthiobiotin from streptavidin (Hirsch et al., 2002). It is desirable to perform downstream processing immediately following biotin-mediated native elution. If not, the sample should be aliquoted and immediately flash frozen in liquid nitrogen for future use.

  6. Downstream processing: Microtiter plate fluorescence assay and gel staining assays
    1. Load 200 μl of biotin eluate into a 96-well black wall, clear bottom microtiter plate and measure fluorescence emission.
    2. To estimate the purity, expression level and integrity of the RNA fraction of the Mango pulldown, phenol chloroform extract 20 μl of the biotin eluate, together with equivalent volumes of input, flow through and wash. Perform denaturing 5% PAGE with TBE as described elsewhere. If the RNA is well expressed, SYBR staining can be used to visualize both the RNA of interest and judge levels of RNA contamination. If the RNA expression levels are low, a Northern blot can be used to judge RNA integrity and expression levels.

  7. Size Exclusion Chromatography (SEC) of streptavidin-purified Mango complex
    As the biotin-eluted RNP complex is highly fluorescent (while it is bound to TO1-Desthiobiotin), it can be further purified with secondary purification techniques such as SEC (Panchapakesan et al., 2017). For SEC purification, load onto a size exclusion column containing SEC resin appropriate for the expected molecular size of the RNP complex being purified. To purify the Mango-tagged 6S RNA in complex with bacterial RNAP, we used a Superdex 200 column (24 ml, 16 mm diameter) using HK buffer at 0.1 ml/min flow rate. In this example, 380 μl of the biotin extract was loaded onto the column. The column was run at 4 °C and 500 μl fractions were collected in silanized plastic tubes.
    1. Fraction analysis: 200 μl from each fraction was placed into a 96-well black wall, clear-bottom microtiter plate, and fluorescence measured using a SpectraMax M5 fluorescence plate reader (settings as previously described). Note that RNA Mango fluorescence is temperature sensitive, maximum fluorescence being obtained at lower temperatures (Jeng et al., 2016). Hence, if the signal to noise ratio is lower at RT, the signal can in principle be improved by measuring fluorescence at 4 °C provided that condensation can be avoided.
    2. Pool the fluorescent fractions of interest. The purified RNP complexes can be analyzed using a variety of techniques such as Mass Spec analysis, Western blot etc. We have used the University of British Columbia Proteomics Core Facility, where samples were analyzed using LC-MS/MS after purification from SDS PAGE gel with success.

Data analysis

For more data analysis such as denaturing gel (Step F2), SEC trace of the purified RNP complex (Step G1), the readers may refer to (Dolgosheina et al., 2014; Panchapakesan et al., 2017).

Notes

  1. KCl is essential for the RNA Mango aptamer to bind TO1-Desthiobiotin, but this binding is only modestly dependent on KCl concentration (Dolgosheina et al., 2014), which can be adjusted as needed (90-150 mM final concentration). However, subsequent generations of Mango (Mango II, III and IV) are much more flexible in optimal KCl concentration range (5-150 mM, Mango III functions optimally at 20 mM KCl) and are functional up to at least 256 mM MgCl2 (Autour et al., 2018).
  2. We have had success performing purification in the absence of RNase and protease inhibitors, but consider adding these to both the BNE and Binding buffers as needed.
  3. Although this protocol extensively deals with purification from bacterial cells, it can also be adopted to work with eukaryotic systems such as yeast cells. If using yeast cells, steps such as native extract preparation (Step B1), binding of extract to beads (Procedure D) and elution (Procedure E) should be changed accordingly.

Recipes

Note: Buffers (prepare as 10x sterile filtered stocks, store at -20 °C or lower).

  1. 10x Bacterial Native Extract (BNE) buffer
    200 mM Tris pH 8.0
    900 mM KCl
    10 mM MgCl2
    Prepare a 100 mM DTT stock and make up 1x BNE buffer to include 1 mM fresh DTT (final) before use
  2. 10x Buffer A
    1,000 mM NaOH
    500 mM NaCl
  3. 10x HEPES KCl (HK) buffer
    150 mM HEPES pH 7.5
    900 mM KCl
  4. 10x Binding buffer
    150 mM HEPES pH 7.5
    900 mM KCl
    750 μg/ml heparin*
    Prepare 1x Binding buffer from HK buffer by adding 1 mM fresh DTT (final) immediately before use. As many RNA binding proteins are positively charged, heparin (a negatively charged sulfated carbohydrate) may be added as a cheap competitor to inhibit nonspecific RNP complex formation
  5. 10x Biotin elution buffer
    200 mM Biotin in 10x Binding buffer
  6. LB media
    10 g tryptone
    5 g yeast extract
    10 g NaCl
    Autoclave prior to use

Acknowledgments

PJU acknowledges an NSERC Discovery operating grant. The authors declare no conflicts of interest or competing interests.

References

  1. Autour, A., S, C. Y. J., A, D. C., Abdolahzadeh, A., Galli, A., Panchapakesan, S. S. S., Rueda, D., Ryckelynck, M. and Unrau, P. J. (2018). Fluorogenic RNA Mango aptamers for imaging small non-coding RNAs in mammalian cells. Nat Commun 9(1): 656.
  2. Dolgosheina, E. V., Jeng, S. C., Panchapakesan, S. S., Cojocaru, R., Chen, P. S., Wilson, P. D., Hawkins, N., Wiggins, P. A. and Unrau, P. J. (2014). RNA mango aptamer-fluorophore: a bright, high-affinity complex for RNA labeling and tracking. ACS Chem Biol 9(10): 2412-2420.
  3. Hirsch, J. D., Eslamizar, L., Filanoski, B. J., Malekzadeh, N., Haugland, R. P., Beechem, J. M. and Haugland, R. P. (2002). Easily reversible desthiobiotin binding to streptavidin, avidin, and other biotin-binding proteins: uses for protein labeling, detection, and isolation. Anal Biochem 308(2): 343-357.
  4. Jeng, S. C., Chan, H. H., Booy, E. P., McKenna, S. A. and Unrau, P. J. (2016). Fluorophore ligand binding and complex stabilization of the RNA Mango and RNA Spinach aptamers. RNA 22(12): 1884-1892.
  5. Leontis, N. B. and Westhof, E. (2001). Geometric nomenclature and classification of RNA base pairs. RNA 7(4): 499-512.
  6. Panchapakesan, S. S. S., Ferguson, M. L., Hayden, E. J., Chen, X., Hoskins, A. A. and Unrau, P. J. (2017). Ribonucleoprotein purification and characterization using RNA mango. RNA 23(10): 1592-1599.
  7. Panchapakesan, S. S., Jeng, S. C. and Unrau, P. J. (2015). RNA complex purification using high-affinity fluorescent RNA aptamer tags. Ann N Y Acad Sci 1341: 149-155.
  8. Trachman, R. J., 3rd, Demeshkina, N. A., Lau, M. W. L., Panchapakesan, S. S. S., Jeng, S. C. Y., Unrau, P. J. and Ferre-D’Amare, A. R. (2017). Structural basis for high-affinity fluorophore binding and activation by RNA mango. Nat Chem Biol 13(7): 807-813.

简介

描述了使用RNA Mango进行RNA-蛋白质复合物的RNA纯化的天然纯化策略。 RNA芒果适体首先被基因工程改造成感兴趣的RNA。 然后将从清除的细胞天然提取物获得的含有RNA复合物的复合物固定在TO1-Desthiobiotin饱和的链霉亲和素琼脂糖珠上。 洗涤珠粒以去除非特异性复合物,然后通过向珠粒中加入游离生物素来洗脱含RNA芒果的复合物。 由于洗脱的复合物是天然的和荧光的,所以可以容易地添加第二纯化步骤如尺寸排阻色谱,并且通过监测荧光追踪纯化的复合物。 通过这种两步纯化策略产生的高纯度天然复合物可以用于进一步的生物化学表征。

【背景】目前的RNA标签受限于诸如差K ,大尺寸,潜在的生物学干扰或缺乏固有荧光的限制(Panchapakesan等人, 2015年)。 RNA芒果很小,可以简单地整合到茎环结构中,特别是GNRA tetraloops中,它具有生物耐受性,并且最重要的是对其噻唑橙基(TO1)配体TO1-Desthiobiotin(TO1-Dtb)具有高亲和力。 。这允许芒果标记的复合物容易结合并在链霉抗生物素蛋白珠上洗涤(总结在图1中)。芒果:TO1-Desthiobiotin复合物是高度荧光的,可通过加入生物素从链霉亲和素珠上洗脱。芒果标记的天然复合物的荧光允许使用另外的纯化步骤来获得高度纯化的天然复合物。

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图1.从天然提取物中纯化芒果标记的RNA-蛋白质复合物设计RNA构建物(程序A,图2),使得芒果标记物位于生物学相容位置。在该构建体的细菌表达之后,制备含有芒果标记的RNA(芒果标签以红色突出显示)的天然提取物(程序B)。然后将芒果标记的RNA和RNA复合物与用TO1-脱硫生物素(Dtb,方法C)衍生化的链霉抗生物素蛋白珠(含S的黑色圆圈)结合。 TO1-Dbt的噻唑橙部分显示为紫色,一旦被芒果结合(亮红色突出显示),就变成高度荧光。然后在天然条件下彻底洗涤结合的复合物(程序D)。洗涤后,可以通过加入生物素在天然条件下洗脱荧光芒果标记的复合物(步骤E)。包括进一步纯化步骤的下游分析可以简单地实施(程序F和G)。

关键字:RNA Mango, TO1脱硫生物素, RNP复合物纯化, 荧光团, 非变性纯化, 非编码RNA, RNA-蛋白质复合物

材料和试剂

  1. 低保留移液器吸头(Fisher Scientific,产品目录号:02-717-134,02-717-143,02-707-511)
  2. 预润滑的微量离心管(Corning,Costar ,产品目录号:3206,3207)
  3. 96孔黑色底壁,底部清澈,无结合,无菌,Greiner Bio-One微量滴定板(Greiner Bio One International,目录号:655906)
  4. Tricorn XK 16/40 SEC柱(GE Healthcare,目录号:28988938)
  5. 带有RNA Mango标签RNA的细菌细胞(大肠杆菌)。
  6. 液氮
  7. TO1-PEG3-Desthiobiotin(ABM,目录号:G956)
  8. 高容量链霉亲和素琼脂糖珠(Thermo Fisher Scientific,Thermo Scientific TM,目录号:20357)
  9. Superdex 200树脂(GE Healthcare,目录号:17104301)
  10. SYBR Green II RNA凝胶染色剂(Thermo Fisher Scientific,Invitrogen TM,目录号:S7564)
  11. 19:1丙烯酰胺:Bis 40%(Thermo Fisher Scientific,目录号:AM9022)
  12. N,N,N',N' - 四甲基乙二胺(TEMED)(Sigma-Aldrich,目录号:T9281)
  13. 过硫酸铵(APS)(Bio-Rad Laboratories,目录号:1610700)
  14. 硼酸(ACP Chemicals,目录号:B2940)
  15. EDTA(ACP Chemicals,目录号:E4320)
  16. Tris碱(Fisher Scientific,目录号:BP152)
  17. Tris-HCl(Sigma-Aldrich,Roche Diagnostics,目录号:10812846001)
  18. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9541)
  19. 氯化镁(MgCl 2)(Sigma-Aldrich,目录号:M8266)
  20. DTT(Sigma-Aldrich,Roche Diagnostics,目录号:10197777001)
  21. 氢氧化钠(NaOH)(VWR,BDH,目录号:BDH9292)
  22. 氯化钠(NaCl)(ACP化学品,目录号:S2830)
  23. HEPES(Sigma-Aldrich,目录号:H3375)
  24. 肝素(Sigma-Aldrich,目录号:H0777-25KU)
  25. 生物素(Sigma-Aldrich,目录号:B4501)
  26. 酵母提取物(Fisher Scientific,目录号:BP1422)
  27. 胰蛋白胨(Fisher Scientific,目录号:BP1421)
  28. 氢氧化钾(KOH)(VWR,BDH,目录号:BDH9262)
  29. 10x细菌天然提取物( BNE )缓冲液(见食谱)
  30. 10倍缓冲 A (见食谱)
  31. 10x HEPES KCl( HK )缓冲液(见食谱)
  32. 10x 绑定缓冲区(请参阅食谱)
  33. 10x生物素洗脱缓冲液(见食谱)
  34. LB 媒体(请参阅食谱)

设备

  1. 标准的个人防护设备
  2. 移液器
  3. 蠕动泵
  4. 馏分收集器和适当的收集管
  5. SpectraMax M5荧光读板器(Molecular Devices,型号:SpectraMax M5)
  6. 温控室(保持在4°C)
  7. 制备离心机,微量离心机和台式Picofuge
  8. 用于1.5毫升管的管旋转器
  9. 聚丙烯酰胺凝胶运行设备
  10. 法国媒体
  11. Sorvall RC6 +离心机(Thermo Fisher Scientific,Thermo Scientific TM,型号:Sorvall TM RC 6 Plus)
  12. Sorvall SLA-1500转子(Thermo Fisher Scientific,Thermo Scientific TM,目录号:SLA-1500)
  13. NanoDrop
  14. 高压灭菌器

程序

  1. 构建设计
    1. RNA Mango的单个拷贝通常足以用于下拉分析,并且可以插入到既不存在干扰感兴趣的RNA功能的预先存在的茎环中(图2 (Panchapakesan

      图2.生物相容性芒果-I标签的设计A.使用Leontis-Westhof命名法(Leontis和Westhof,2001)描述了一般的GNRA四环结构。 B.芒果I的结构(由Autour等人修改的图(<2018年)>)。通过用感兴趣的RNA的茎序列代替紫色突出的N-N'茎,可以将芒果掺入到自然发生的茎环结构中。在芒果I中,GAAA四环状基序(蓝色)通过侧翼包围核心G-四链体(3层:T1,T2,T3),使得芒果I核适合GAA / A,其中'/'是芒果核心插入点。因此,只要芒果不干扰四环素的生物功能,芒果I可以以类似的方式插入预先存在的GNRA四环结构中。

    2. 芒果标记的RNA应该针对芒果标签的功能以及感兴趣的RNA进行测试。芒果标签的功能可以通过测量感兴趣的芒果标签RNA的荧光并将其与体外转录的RNA芒果(Panchapakesan 等<2017 )。通常,在1x结合缓冲液中制备1μMRNA溶液以及2倍过量的TO1-Dtb配体。在室温下孵育5分钟后,可以使用荧光计进行荧光测定。对于SpectraMax M5荧光计,以下激发和发射设置对于分析RNA芒果介导的荧光是最佳的:在495nm激发;发射535纳米PMT:中等,截止515纳米 - 底部读取。在上述设置下使用SpectraMax M5,可以可靠地检测低至5 nM的RNA芒果浓度。用户应优化激发和发射波长,以尽量减少所用特定荧光计激发光源的干扰。&nbsp;

  2. 天然提取物制备
    1. 具有RNA Mango标记的感兴趣的RNA的细菌细胞(大肠杆菌)应该在适当的生长条件下生长以最大限度地形成所需的RNP复合物。我们通常在1L LB培养基中过夜培养细胞。
    2. 然后使用Sorvall SLA-1500转子,在冷冻Sorvall RC6 +离心机中将细胞在3800×gg(RCF)下沉淀10分钟。然后将得到的细胞沉淀物重新悬浮在20ml预先冰冻的BNE缓冲液中。通过在1000 psi下两次通过4°C法式压力机传代裂解细胞。如果法国报刊不可用,那么可以使用替代细胞破碎技术,如超声处理或酶法如溶菌酶消化。然后通过在13,200×g(RCF)下在4℃下离心10分钟来澄清该粗裂解物。澄清的裂解物在液氮中快速冷冻并储存在-80℃直到准备使用。对于其他生物体,包括酵母,应使用适当的天然提取物制备方案。
    3. 由于与TO1-Desthiobiotin的结合需要KCl(Dolgosheina等人,2014; Jeng等人,2016; Panchapakesan 等,2017)。
      在步骤B2和B3中处理所有材料

  3. TO1-Desthiobiotin链霉亲和素琼脂糖珠粒制备
    准备适量的链霉亲和素琼脂糖珠浆液。使用7ml细菌提取物和700μl链霉亲和素琼脂糖珠浆液,用42纳摩尔的TO1-Desthiobiotin进行衍生化,我们在过表达6S调节RNA时获得了高质量的结果(Dolgosheina等人,2014; Panchapakesan 等人,2017)。根据实验条件缩放下面的协议。

    1. 小心地重新悬浮链霉亲和素琼脂糖珠浆

    2. 在1.5 ml Eppendorf管中吸取700μl链霉亲和素琼脂糖珠
    3. 使用标准实验室旋转器在室温(RT)下用1ml缓冲液A(每次2分钟)洗涤两次。可以在Picofuge中简单离心管以从珠中除去多余的液体。
    4. 使用1ml HK缓冲液洗涤珠3次,每次洗涤2分钟,再次使用旋转器。
    5. 对于每100μl链霉亲和素琼脂糖珠,在100μlHK缓冲液中与6纳摩尔TO1-Desthiobiotin孵育。在RT上在旋转器上孵育15分钟。注意前和后的荧光团溶液的OD 500(λ500 = 63,000M -1 -1 cm -1)在与珠粒结合后使用NanoDrop估计结合的荧光团的量。 TO1-Desthiobiotin的90%以上应该受到约束。由于荧光团结合,珠子的颜色现在变成橙色。不同的珠子可能具有不同的结合能力,如果需要,应该凭经验确定。
    6. 用1ml的 HK 缓冲液清洗残余的未结合的荧光团。

  4. 提取物与制备的琼脂糖珠的结合
    由于该过程的最终目标是在天然条件下纯化RNP复合物,所以应注意不要在纯化的每个步骤中变性复合物,即珠结合,洗涤和洗脱。由于RNA Mango需要KCl以正确折叠并与TO1-目标生物素配体结合(Dolgosheina等人,2014; Jeng等人,2016; Trachman III等人, et al。,2017),KCl应包括在所有结合,洗涤和洗脱步骤中。缓冲区的其他组件可以根据实验需要进行修改。
    1. 在RT下将7ml细菌提取物结合到珠上。在RT上孵育15分钟(在酵母提取物的情况下(Panchapakesan等人,2017),在4℃下进行结合以确保RNP复合物的稳定)。如果需要,可以通过向提取物中加入 BNE 或结合缓冲液来稀释提取物。
    2. 用1ml强结合缓冲液洗涤两次,每次循环2分钟。选择的洗涤次数取决于预期的RNP综合体的稳定性。同样,如果需要优化,每次洗涤的温度(4-37°C)和时间(2-15分钟)都可以进行显着调整。

  5. 生物素介导的本地洗脱
    在旋转器上使用20mM生物素洗脱缓冲液以500μl体积在37℃洗脱(Panchapakesan等人,2017)20分钟,因为生物素有效地从链霉抗生物素蛋白中取代脱硫生物素(Hirsch et al。,2002)。在生物素介导的天然洗脱之后立即进行下游加工是合乎需要的。如果不是,则应将样品分装并立即在液氮中快速冷冻备用。

  6. 下游处理:微量滴定板荧光测定和凝胶染色测定
    1. 将200μl生物素洗脱液加载到96孔黑色壁上,清除底部微量滴定板并测量荧光发射。
    2. 为了估计芒果下拉的RNA部分的纯度,表达水平和完整性,苯酚氯仿提取20μl生物素洗脱液以及等量的输入,流过并洗涤。如其他地方所述用TBE进行变性5%PAGE。如果RNA的表达良好,SYBR染色可以用来显示感兴趣的RNA和判断RNA污染水平。如果RNA表达水平低,Northern印迹可以用来判断RNA的完整性和表达水平。

  7. 链霉亲和素纯化的芒果复合物的尺寸排阻色谱(SEC)
    由于生物素洗脱的RNP复合物是高度荧光的(虽然它与TO1-Desthiobiotin结合),但它可以用二次纯化技术如SEC进一步纯化(Panchapakesan等人,2017)。对于SEC纯化,加载到含有SEC树脂的大小排阻柱上,所述SEC树脂适合被纯化的RNP复合物的预期分子大小。为了纯化与细菌RNAP复合的芒果标记的6S RNA,我们使用Superdex 200柱(24ml,16mm直径),使用0.1ml / min流速的HK缓冲液。在这个例子中,380微升生物素提取物被加载到色谱柱上。该柱在4℃下运行,500μl级分收集在硅烷化塑料管中。
    1. 馏分分析:将来自各馏分的200μl置于96孔黑色壁,透明底部微量滴定板中,并使用SpectraMax M5荧光板读数仪(如前所述设定)测量荧光。请注意,RNA芒果荧光是温度敏感的,在较低温度下获得最大荧光(Jeng et al。,2016)。因此,如果RT下的信噪比较低,则原则上可以通过在4°C下测量荧光来改善信号,前提是可以避免冷凝。
    2. 汇集感兴趣的荧光部分。可以使用多种技术如质谱分析,蛋白质印迹等来分析纯化的RNP复合物。我们使用了不列颠哥伦比亚大学蛋白质组学核心设施,使用LC-MS / MS分析样品,经SDS PAGE凝胶纯化后成功。

数据分析

对于更多的数据分析,如变性凝胶(步骤F2),纯化的RNP复合物的SEC迹线(步骤G1),读者可参考(Dolgosheina等人,2014; Panchapakesan等人,2017)。

笔记

  1. KCl对于RNA Mango适体与TO1-Desthiobiotin结合是必不可少的,但这种结合仅适度依赖于KCl浓度(Dolgosheina等人,2014),可根据需要进行调整(90-150 mM终浓度)。然而,随后几代芒果(芒果II,III和IV)在最佳KCl浓度范围内(5-150 mM,Mango III在20 mM KCl下最佳起作用)灵活得多,并且功能性高达至少256 mM MgCl )。
  2. 我们在没有RNA酶和蛋白酶抑制剂的情况下进行纯化时已经取得了成功,但可以根据需要考虑将这些添加到缓冲液和缓冲液中。
  3. 尽管该方案广泛涉及从细菌细胞纯化,但它也可以用于与真核系统如酵母细胞一起工作。如果使用酵母细胞,应相应地改变诸如天然提取物制备(步骤B1),提取物与珠粒(步骤D)和洗脱(步骤E)的结合等步骤。

食谱

注意:缓冲液(准备为10倍无菌滤过的原液,储存在-20°C或更低)。

  1. 10x细菌天然提取物( BNE )缓冲液
    200 mM Tris pH 8.0
    900毫摩尔KCl
    10mM MgCl 2 2/2 准备100 mM DTT储备液并在使用前组成1x BNE 缓冲液以包含1 mM新鲜DTT(最终)
  2. 10x缓冲区 A
    1,000 mM NaOH
    500 mM NaCl
  3. 10倍HEPES KCl( HK )缓冲液
    150 mM HEPES pH 7.5
    900毫摩尔KCl
  4. 10x 绑定缓冲区
    150 mM HEPES pH 7.5
    900毫摩尔KCl
    750μg/ ml肝素*
    在使用前立即加入1mM新鲜DTT(最终),从 HK 缓冲液中准备1x Binding 缓冲液。由于许多RNA结合蛋白带正电,肝素(带负电荷的硫酸化碳水化合物)可作为廉价竞争者加入,以抑制非特异性RNP复合物的形成。
  5. 10倍生物素洗脱缓冲液

    缓冲液中含有200 mM生物素
  6. LB 媒体
    10克胰蛋白胨
    5克酵母提取物
    10克NaCl
    使用前的高压灭菌器

致谢

PJU承认NSERC发现运营资助。作者声明不存在利益冲突或利益冲突。

参考

  1. Autour,A.,S。C.Y.J.,A,D.C.,Abdolahzadeh,A.,Galli,A.,Panchapakesan,S.S.S.,Rueda,D.,Ryckelynck,M.and Unrau,P.J。(2018)。 荧光RNA芒果核酸适体用于哺乳动物细胞中非编码小RNA的成像 Nat Commun 9(1):656.
  2. Dolgosheina,E.V.,Jeng,S.C.,Panchapakesan,S.S.,Cojocaru,R.,Chen,P.S.,Wilson,P.D.,Hawkins,N.,Wiggins,P.A。和Unrau,P.J。(2014)。 RNA芒果适体 - 荧光团:一种用于RNA标记和跟踪的明亮,高亲和力复合物。 ACS Chem Biol 9(10):2412-2420。
  3. Hirsch,J.D.,Eslamizar,L.,Filanoski,B.J.,Malekzadeh,N.,Haugland,R.P.,Beechem,J.M。和Haugland,R.P。(2002)。 易于逆转的脱硫生物素与链霉亲和素,抗生物素蛋白和其他生物素结合蛋白的结合:用于蛋白质标记,检测和隔离。 Anal Biochem 308(2):343-357。
  4. Jeng,S.C.,Chan,H.H.,Booy,E.P.,McKenna,S.A。和Unrau,P.J。(2016)。 荧光配体结合和RNA Mango和RNA Spinach核酸适体的复杂稳定化 RNA 22(12):1884-1892。
  5. Leontis,N.B.和Westhof,E.(2001)。 RNA碱基对的几何命名和分类 RNA 7(4):499-512。
  6. Panchapakesan,S.S.S.,Ferguson,M.L.,Hayden,E.J.,Chen,X.,Hoskins,A.A。和Unrau,P.J。(2017)。 使用RNA芒果进行核糖核蛋白纯化和表征 RNA 23 (10):1592-1599。
  7. Panchapakesan,S. S.,Jeng,S.C。和Unrau,P.J。(2015)。 使用高亲和力荧光RNA适体标签进行RNA复合物纯化 Ann NY Acad Sci1341:149-155。
  8. Trachman,R.J.,3rd,Demeshkina,N.A.,Lau,M.W.L.,Panchapakesan,S.S.S.,Jeng,S.C.Y.,Unrau,P.J。和Ferre-D'Amare,A.R。(2017)。 RNA芒果高亲和性荧光素结合和活化的结构基础 Nat Chem Biol 13(7):807-813。
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引用:Panchapakesan, S. S., Jeng, S. C. and Unrau, P. J. (2018). Purification of RNA Mango Tagged Native RNA-protein Complexes from Cellular Extracts Using TO1-Desthiobiotin Fluorophore Ligand. Bio-protocol 8(7): e2799. DOI: 10.21769/BioProtoc.2799.
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