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May 2019
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Confocal and Super-resolution Imaging of RNA in Live Bacteria Using a Fluorogenic Silicon Rhodamine-binding Aptamer
含氟硅-罗丹明适配体用于活性菌中RNA的激光共聚焦高分辨率成像   

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Abstract

Genetically encoded light-up RNA aptamers have been shown to be promising tools for the visualization of RNAs in living cells, helping us to advance our understanding of the broad and complex life of RNA. Although a handful of light-up aptamers spanning the visible wavelength region have been developed, none of them have yet been reported to be compatible with advanced super-resolution techniques, mainly due to poor photophysical properties of their small-molecule fluorogens. Here, we describe a detailed protocol for fluorescence microscopy of mRNA in live bacteria using the recently reported fluorogenic silicon rhodamine binding aptamer (SiRA) featuring excellent photophysical properties. Notably, with SiRA, we demonstrated the first aptamer-based RNA visualization using super-resolution (STED) microscopy. This imaging method can be especially valuable for visualization of RNA in prokaryotes since the size of a bacterium is only a few times greater than the optical resolution of a conventional microscope.

Keywords: Light-up aptamer (Light-up适配体), Silicon rhodamine (硅-罗丹明), RNA imaging (RNA成像), STED microscopy (受激发射损耗荧光显微镜), Confocal fluorescence microscopy (荧光共聚焦显微镜), Live cell imaging (活细胞成像), SiRA aptamer (SiRA适配体)

Background

The visualization of specific RNA molecules by fluorescence microscopy has become invaluable during the past two decades to extend our knowledge of RNA function within cells in a spatiotemporal manner (Tyagi, 2009; Xia et al., 2017). Due to the lack of inherently fluorescent RNAs, development of fluorogenic RNA labeling tools for live-cell imaging and their adaptation to state-of-the-art microscopes–especially to super-resolution microscopes–is imperative. Super-resolution microscopy (SRM) is particularly attractive for imaging RNAs in prokaryotic systems since a bacterium is very small (~2.5 μm long, ~0.5-1 μm wide) and the resolution of a standard fluorescence microscope is restricted to ~200-300 nm due to the diffraction limit of light (Reshes et al., 2008; Nienhaus and Nienhaus, 2016). Although a plethora of tools have been developed to fluorescently label an RNA of interest (ROI), their live-cell visualization using SRM (resolution ~20-50 nm) is still challenging (Alexander and Devaraj, 2017), mainly due to the stringent requirements on the photophysical properties of the fluorophores needed for live-cell SRM. These properties include high photostability and brightness, cellular membrane permeability, water solubility, absorption and emission wavelengths preferentially in the far-red and near-infrared (NIR) region, on-off switching capability and a sufficient signal-to-background ratio during imaging (Wang et al., 2019). Thus, there is a strong need for novel RNA reporter systems that are compatible with SRM.

An especially attractive approach to visualize RNAs in live cells is the use of genetically encoded fluorescence light-up aptamers, which are short oligonucleotides that bind small, conditionally fluorescent probes with high affinity and are developed using systematic evolution of ligands by exponential enrichment (SELEX) (Holeman et al., 1998; Ouellet, 2016). Tagged to the ROI, the aptamers can be visualized due to a fluorescence increase upon small molecule binding (Figure 1). Several fluorescence enhancing RNA aptamers spanning the visible region of the spectrum have recently been developed and proven to be powerful tools for RNA labeling (Bouhedda et al., 2017; Neubacher and Hennig, 2019).

The famous Spinach, Broccoli, Mango, Corn aptamers and their improved variants bind to fluorophores with low fluorescence quantum yield in solution and become fluorescent due to the restriction of intramolecular movements upon aptamer binding (Figure 1A) (Paige et al., 2011; Filonov et al., 2014; Dolgosheina et al., 2014; Song et al., 2017). Other prominent examples are SRB-2, DNB, BHQ and Riboglow aptamers that disrupt a quencher-dye conjugate upon binding to either the dye or the quencher moiety, thereby generating fluorescence enhancement (Figure 1B) (Murata et al., 2011; Sunbul and Jäschke, 2013 and 2018; Arora et al., 2015; Braselmann et al., 2018). Most of these aptamer-ligand pairs, however, do not absorb and emit in the far-red or NIR region. Although MG, Riboglow, Mango TO-3 and the recently reported DIR-pro (which has not been reported for RNA labeling) are excited in the far-red, their fluorophores show either low photostability and brightness, phototoxicity or are hardly cell permeable (Tan et al., 2017; Autour et al., 2018; Yerramilli and Kim, 2018).

Prompted by these shortcomings, we recently developed a novel light-up RNA aptamer that binds to 3-carboxy silicon rhodamine (SiR) (Wirth et al., 2019). SiRs are an exciting new fluorophore class in the field of protein labeling for SRM because they are photostable, NIR-emitting fluorophores that change their equilibrium between the non-colored spirolactone and the fluorescent zwitterion in response to their environment (Figure 1C) (Lukinavičius et al., 2013). This property is responsible for their high cell permeability and fluorogenic behavior and contributes to the popularity of SiR dyes and their derivatives in the last decade (Wang et al., 2019). Applying SELEX on a combinatorial RNA library with a diversity of ~2 x 1015, we discovered a 50 nt-minimal aptamer, SiRA, which binds the target SiR 1 with a KD of 430 nM and shows a significant fluorescence turn-on upon dye binding (Figure 2). This finding introduces a new concept of fluorogenicity to the field of aptamer-based RNA imaging, namely environment-dependent intramolecular spirocyclization (Figure 1C). SiRA is remarkably resistant to photobleaching and constitutes the brightest far-red light up aptamer system known to date (quantum yield 0.98; extinction coefficient 86,000 M-1·cm-1). Using the SiRA system, we visualized the expression of RNAs in bacteria in no-wash live-cell imaging experiments (Wirth et al., 2019). The innovation in this protocol is that we can use SiRA’s excellent photophysical properties for stimulated emission depletion (STED) SRM of mRNA in live bacteria. To date, SiRA is the only light-up aptamer system proven to work in SRM applications.



Figure 1. Fluorogenicity concepts of light-up RNA aptamers used for RNA imaging. A. A fluorophore with low fluorescence quantum yield due to molecular motions in solution (OFF) becomes highly fluorescent upon aptamer binding due to the restriction of intramolecular movements (ON). B. The contact-quenched fluorophore-quencher conjugates (OFF) light up upon binding to either the fluorophore or the quencher moiety (ON). Here, an example of a light-up system using a fluorophore binding aptamer was shown. C. In solution, SiRs can favor the non-colored spirolactone (OFF). The change in environment upon aptamer binding causes a shift of the equilibrium to the fluorescent zwitterion (ON).


Figure 2. The SiRA aptamer and its properties. A. The predicted secondary structure of the 50-nt SiRA aptamer evolved during the selection process (important features are marked in color). B. Structure of the fluorogenic dye 1 used in this protocol, which binds to SiRA with nanomolar affinity. C. Normalized absorption and emission spectra of free SiR 1 and the [SiRA*1] complex.

In the following protocol, we describe the preparation of E. coli bacteria for live-cell confocal and STED imaging of GFP-mRNA in detail. We herein use a plasmid-based expression system and tag the GFP-mRNA with tandem repeats of the novel SiRA aptamer (SiRA5) at the 3′ untranslated region (UTR). This protocol can be adapted to visualize any plasmid expressed target RNA; however, the expression levels and the half-life of RNAs need to be kept in mind. Further optimization of the protocol might be required for specific applications.

Materials and Reagents

  1. Pipette tips (Sarstedt, catalog numbers: 70.113 0, 70.760.002 , 70.762 )
  2. Microcentrifuge tubes 1.5 ml (Sarstedt, catalog number: 72.690.001 )
  3. 15 μ-Slide 8-well uncoated, glass bottom #1.5 polymer coverslip (Ibidi GmbH, catalog number: 80827 )
  4. High clarity PP conical centrifuge tube 15 ml (Falcon®, catalog number: 352096 )
  5. High clarity PP conical centrifuge tube 50 ml (Falcon®, catalog number: 352070 )
  6. Medical Millex-GS syringe filter unit, 0.22 μm (EDM Merck, catalog number: SLGSM33SS )
  7. BD Discardit disposable syringes 10 ml (Becton Dickinson, catalog number: 300296 )
  8. PYREX® Media bottles, graduated, Corning®, 1,000 ml (VWR, PYREX®, catalog number: 1395-10L )
  9. FluoSpheresTM Carboxylate-Modified Microspheres, 0.04 μm, dark red fluorescent (660/680), 5% solids, azide free (Thermo Fisher Scientific, catalog number: F8789 )
  10. Gold beads (GC80, BB International, catalog number: EM. GC80 )
  11. E. coli BL21 StarTM (DE3) Invitrogen competent E. coli strains (Thermo Fisher, catalog number: C601003 ), store at -80 °C
  12. pET-GFP expression plasmid (pET His6 GFP TEV LIC cloning vector, 1 GFP) (Addgene, Plasmid number: 29663), store at -20 °C
  13. pET-GFP-SiRA5 expression plasmid, store at -20 °C
    The pET-GFP-SiRA5 plasmid used in this protocol is not commercially available. Generally tandem repeats of an aptamer of choice (depending on the target RNA and expression level), can directly be fused to the ROI (in this case GFP mRNA) using proper restriction sites in the 3′ UTR (here: SalI and XhoI fast digest restriction enzymes, Thermo ScientificTM, catalog numbers: FD0644 and FD0695 , respectively) as previously described in the literature (Sunbul et al., 2018). In this study, we inserted five repeats of the SiRA aptamer with interspersed linker nucleotides according to Figure 3.


    Figure 3. Excerpt of the pET-GFP-SiRA5 bacterial expression plasmid used for the visualization of GFP mRNA in this protocol. lacO: lac operator, rbs: ribosomal binding site, ROI: RNA of interest, P: promoter, T: terminator, SalI and XhoI: Restriction sites, stop: Stop codon for RNA translation.

    The following sequences show an excerpt of the cloned expression vector (pET-GFP-SiRA5) as well as the pET-GFP used in this study.

    pET-GFP-SiRA5:
    ...CGAGGAAACCTGTACTTCCAATCCAATATTGGAAGTGGATAACGGATCCGAATTCGAGCGCCGTCGACAGGCCCACCGGGTTTGAAAACCTGGCTGCTTCGGCAGTTGTATCCTTTGGGCCTAAGAGAGACCACCGGGTTTGAAAACCTGGCTGCTTCGGCAGTTGTATCCTTTGGTCTCAATAACCAGCCACCGGGTTTGAAAACCTGGCTGCTTCGGCAGTTGTATCCTTTGGGCCTAAGAGAGACCACCGGGTTTGAAAACCTGGCTGCTTCGGCAGTTGTATCCTTTGGTCTCAATAACCAGCCACCGGGTTTGAAAACCTGGCTGCTTCGGCAGTTGTATCCTTTGGCTGGCTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTG…
    pET-GFP:
    ...CGAGGAAACCTGTACTTCCAATCCAATATTGGAAGTGGATAACGGATCCGAATTCGAGCGCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTG…
    black     SiRA (3-nt stem loop)
    grey     Variable Stem Loop or Spacer Region
    blue     SalI Restriction Site
    pink     XhoI Restriction Site

  14. Autoclaved LB medium, Lennox (Carl Roth®, catalog number: X964.4 )
  15. LB Broth with agar, Lennox (Sigma-Aldrich, catalog number: L2897 )
  16. Kanamycin sulfate from Streptomyces kanamyceticus (Sigma-Aldrich, catalog number: K1377 ), prepare 1 M aqueous solution, filter-sterilize and store at -20 °C
  17. Isopropyl-β-D-thio-galactopyranoside (IPTG) (Sigma-Aldrich, catalog number: I5502 ), prepare 1 M aqueous solution, filter-sterilize and store at -20 °C
  18. Potassium hydroxide pellets EMPLURA® (Sigma-Aldrich, catalog number: 1050121000 ), prepare 10 M aqueous solution
  19. Poly-D-lysine hydrobromide (Sigma-Aldrich, catalog number: P7886 ), prepare 2.5 mg/ml in DPBS, store at 4 °C
  20. Dulbecco's Phosphate Buffered Saline (DPBS) (Sigma-Aldrich, catalog number: D8537 )
  21. MilliQ water
  22. Silicon rhodamine based fluorogenic dye, 100 μM solution in DMSO
    The fluorogenic dye used in this protocol is not commercially available. Therefore, it has to be synthesized and purified by HPLC as described in the literature (Wirth et al., 2019).
    Note: The HPLC purification has to be carried out with great care, since the fluorescence of remaining impurities can decrease the signal-to-background ratio during the imaging experiment.
  23. M9 Broth (Fluka Sigma-Aldrich, catalog number: 63011 )
  24. Magnesium sulfate monohydrate (Sigma-Aldrich, catalog number: 434183 ), prepare 1 M aqueous solution and autoclave
  25. D-(+)-Glucose (Sigma-Aldrich, catalog number: G8270 ), prepare 40% (w/v) aqueous solution and filter-sterilize
  26. Magnesium chloride hexahydrate (Sigma-Aldrich, catalog number M2670 ), prepare 1 M aqueous solution and autoclave
  27. Calcium chloride dihydrate (Sigma-Aldrich: catalog number: 1725801000 ), prepare 1 M aqueous solution and autoclave
  28. Live-cell imaging solution (Thermo Fisher Scientific, catalog number: A14291DJ )
  29. M9 imaging solution (see Recipes)

Equipment

  1. Research® plus pipettes, variable volume 200 and 1,000 μl (Sigma-Aldrich, Eppendorf®, catalog numbers: Z683817 and Z683825 )
  2. Steam Sterilizer (e.g., Varioklav®, model: 155S )
  3. Thermomixer comfort 24 x 1.5 ml (Eppendorf®)
  4. Incubator and shaker (Thermo Fisher Scientific, type 496)
  5. BioPhotometer (Eppendorf®, type 6131)
  6. High speed Micro Centrifuge (Neuation Technology Pvt. Ltd., catalog number: iFuge M12 )
  7. Freezer
  8. Refrigerator
  9. Confocal microscope (Nikon, model: A1R )
    For this protocol, a point scanning confocal microscope with hybrid scanner (galvano/resonant) was used, equipped with a Nikon N Apo 60x NA 1.4 λs OI (WD 0.14 mm, FOV 0.21 x 0.21 mm) objective. For fluorescence excitation, 488 nm (green channel) and 640 nm (red channel) lasers were used. For detection, a high-sensitive GaAsP detector combined with 525/50 nm and 700/75 nm (center wavelength/bandwidth) emission filters for green and red channels, respectively, was used.
  10. STED microscope
    The STED microscope used in this protocol is home-built and has been described in detail (Gao et al., 2017). In addition, a 650-nm long-pass dichroic mirror (ET700SP; Chroma, Bellows Falls, VT) and a time-correlated single photon counting (TCSPC) card (SPC-150, Becker & Hickl GmbH, Berlin, Germany) are needed.
    Note: The experiment in the protocol was carried out with 640 nm excitation and 779 nm depletion; only the vortex phase plate for plain 2D STED imaging was used.

Software

  1. ImageJ Fiji, version 2.0.0. with Java 1.6.0_24
  2. Software confocal microscope: NIS-Elements image acquisition and analysis software (Nikon)
  3. Software STED microscope: Imspector (Max-Planck-Innovation GmhH, Muenchen, Germany)
  4. Matlab (MathWorks, Natick, MA)
  5. OriginPro 2015 32 bit (OriginLab, Northampton, MA)

Procedure

  1. E. coli transformation (Day 1: ~2.5 h followed by over-night incubation)
    Note: We recommend a fresh transformation prior to each imaging experiment to ensure proper mRNA-SiRA expression.
    1. Add 50 ng of the plasmids (pET-GFP and pET-GFP-SiRA5) into each autoclaved microcentrifuge tube and put them on ice.
    2. Thaw chemically competent E. coli BL21 StarTM (DE3) cells for 15 min on ice.
    3. Add 40 μl of E. coli BL21 StarTM (DE3) cells to each tube and incubate for an additional 30 min on ice.
    4. Heat the tubes for 45 s at 42 °C followed by incubation on ice for 2 min.
    5. Add 250 μl of pre-warmed (37 °C) LB medium to each tube and shake them at 650 rpm for 60 min.
    6. For the selection of bacteria carrying the plasmid, streak 20 μl of the bacteria on LB agar plates containing 30 μg/ml of kanamycin.
    7. Incubate the LB agar plates overnight at 37 °C.
      Note: In the morning, place the agar plates at 4 °C and keep them there throughout the day.

  2. Induction of mRNA expression in bacteria (Day 2: 15 min followed by over-night incubation; Day 3: ~ 5 h)
    1. Add 5 ml of LB medium supplemented with 30 μg/ml kanamycin into a 15 ml Falcon and start an overnight culture using a single colony picked from each LB agar plate using a sterile pipette tip.
    2. Incubate the Falcon at 37 °C with vigorous shaking at 170 rpm overnight.
    3. The next morning, measure the optical density (OD600) for a 1:10 dilution of each overnight culture.
    4. Generate a starter culture from the overnight culture. For this, add 10 ml of LB medium supplemented with 30 μg/ml kanamycin in a 50 ml Falcon and add the overnight culture to give an OD600 of 0.05.
    5. Shake the culture at 37 °C and 170 rpm till the OD600 reaches 0.35.
      Note: This usually takes 1.5-2 h.
    6. Induce the expression of GFP mRNA by adding 10 μl of IPTG (1 mM final concentration) and shake the culture for an additional 3 h at 37 °C.
      Note: Currently the SiRA system has only been used for imaging highly overexpressed GFP mRNAs. In order to visualize lower copy numbers, a larger number of tandem repeats can be designed and further optimization of the protocol (e.g., dye concentration) might be necessary.

  3. Preparation of poly-D-lysine coated glass chamber slides (Day 3: ~3 h, in parallel to Steps B5-B6)
    Note: The poly-D-lysine solution for coating needs to be prepared freshly every time just before adding it to the well. The poly-D-lysine coated glass chamber slides, however, can be prepared up to 24 h in advance and stored in the refrigerator at 6 °C. Warming up to RT before use is recommended.
    1. Remove the 8-well ibidi glass slides from the packaging and incubate each well with 200 μl of a 10 M aqueous KOH solution for 5 min.
    2. Remove the KOH solution from the wells very carefully, wash them thoroughly multiple times with 400 μl sterile MilliQ water and air-dry them for 60 min.
    3. Mix 1,000 μl of water with 20 μl of the poly-D-lysine stock (~50 μg/μl final concentration) and add 250 μl of the solution quickly into each well (recipe for 4 wells).
    4. Incubate the wells for 45 min at room temperature.
    5. Wash each well twice with 300 μl sterile MilliQ water and air-dry for at least 30 min prior to the immobilization of the bacteria.

  4. Preparation of E. coli samples for imaging (Day 3: ~1 h)
    Note: For live-cell imaging experiments, we recommend to image up to 2 wells at a time, due to the short mRNA half-life and bacterial metabolism.
    1. Transfer a 200 μl aliquot of each culture (Step B6) to an autoclaved microcentrifuge tube and pellet the cells at 5,600 x g for 2 min at room temperature.
    2. Remove the LB medium and suspend the bacteria in 1 ml of M9 imaging solution (Recipe 1).
    3. Add 200 μl of the cell suspension into each well of the coated 8-well ibidi slide.
    4. Incubate the bacteria for 35 min at room temperature to allow for adhesion to the surface.
    5. Gently wash each well with 400 μl of M9 imaging solution.
    6. Add 300 μl of M9 imaging solution containing 500 nM of the silicon rhodamine fluorogenic dye and directly prepare for imaging.
      Note: Permeation of the dye into the bacterium is quite fast (5-10 min) at 37 °C; therefore, the time for microscope adjustment is sufficient for incubation. If permeation issues occur, prewarming of the dye/M9 imaging solution to 37 °C is recommended.

  5. Imaging GFP-SiRA5 mRNA using a confocal microscope (Day 3: ~1-2 h)
    1. Turn on the 488 nm (green channel) and 640 nm (red channel) lasers for fluorescence illumination of the point scanning confocal microscope and use the 525/50 nm and 700/75 nm emission filters for green and red channel, respectively.
    2. Attach the temperature incubation chamber to the microscope, prewarm to 37 °C and place the 8-well slide with adherent E. coli into the chamber.
    3. Use the bright-field illumination to focus on the immobilized E. coli with the Nikon N Apo 60x NA 1.4 λs OI (WD 0.14 mm, FOV 0.21 x 0.21 mm) objective, starting with the pET-GFP-SiRA5 transformed cells.
    4. Determine a suitable exposure time for the GFP and red channel as well as the correct z-axis. Choose the settings such that you do not saturate the pixels but still obtain the highest fluorescent signal.
    5. Acquire the brightfield and fluorescence images of all channels (Figure 4).


      Figure 4. Example of GFP mRNA imaging in live E. coli using a confocal microscope. A. Schematic of the plasmid used in the imaging experiment. Binding of the fluorophore to the SiRA aptamers generates a fluorescence signal in the red detection channel of the microscope. B. No-wash live-cell imaging of GFP mRNA in live E. coli cells. The cells were transformed with either the GFP plasmid or the GFP-SiRA5 plasmid, and the images taken with the bright field, green (protein expression) and red channel (mRNA detection). Scale bars = 5 µm. C. Zoom-in of the images depicted in Figure 4B. Scale bars = 5 µm.

    6. Repeat Steps E3-E5 with the E. coli cells carrying the control plasmid pET-GFP using the same microscope settings.
    7. For background correction, we use Fiji/Image J image analysis software. Manually pick a surface area without attached E. coli cells and subtract its mean fluorescence signal from the whole image.

  6. (Optional) Adjustment of the STED microscope
    1. Ensure spatial and temporal overlap of the 640-nm excitation and 779-nm depletion beam foci by scanning a gold bead (GC80) with detection in both color channels.
    2. At first, separate the reflected/scattered excitation and depletion light beams into two beams by a 650-nm long-pass dichroic mirror. Then, filter the two beams by band-path filters 780/10 nm (center/width) and 640/10 nm until they are detected by the two avalanche photodiodes (APDs).
      Use the Imspector software to synchronize the scan signal and photon detection intervals, thus generating two-channel images without spectral crosstalk.
      Note: If necessary, the depletion beam needs to be slightly adjusted to ensure that the center of the STED image (with a doughnut-like profile) is precisely overlaid with the excitation image (with a Gaussian profile).
    3. Check the temporal overlap between the excitation and depletion pulses. Therefore, collect the reflected 640-nm and 779-nm light by the gold beads by using a time-correlated single photon counting (TCSPC) card and sort them with respect to the common trigger signal. The delay between the 640 nm excitation and 779 nm pulses can be monitored in the oscilloscope display-module of the TCSPC software.
      Note: For optimal depletion, the delay between the excitation and depletion pulses can be adjusted (in our example ~200 ps) by using a delay box, which routes the trigger output of the depletion laser to the trigger input of the excitation laser.

  7. Imaging dark-red beads (Day 3: ~1 h)
    Note: Images of dark-red beads are taken to determine the resolution of the STED microscope and should be carried out before or in parallel to the sample preparation.
    1. Dilute FluosphereTM 40-nm dark-red beads (660/680) from the stock solution into PBS at a volume ratio of 1:104.
    2. Add 200 μl of the solution to one of the poly-D-lysine coated imaging chambers and incubate for 5 min at room temperature to immobilize the beads on the surface.
    3. Wash the coverslip twice with 250 μl MilliQ water and finally add 250 μl MilliQ water for imaging.
    4. Place the coverslip onto the microscope slide holder.
    5. Adjust the excitation power to 5 μW at the sample and image with the depletion laser at different powers between 0 mW to 150 mW. Scan a rectangular area of 10 x 10 μm2 with 1,024 x 1,024 pixels, covering dozens of individual dark-red beads.
      Note: To minimize photo-bleaching upon multiple scans of the same region, it is recommended to use a fresh region for STED imaging at the different STED laser intensities.
    6. Generate a comparable image between confocal and STED modes (Figure 5A). Therefore, image the same area sequentially by confocal and STED microscopy.
      Note: By changing the depletion power, the effective resolution can be varied, as shown by images of a bead taken at different powers of the depletion beam (Figure 5B).


      Figure 5. Dark-red bead images. A. Confocal (upper left) and STED (lower right) image of fluorescent beads on a glass coverslip, showing the enormous resolution enhancement due to STED imaging. Scale bar = 3 µm. B. Image of an individual bead taken at different powers of the depletion beam.

  8. Evaluation of the STED resolution using dark-red bead images (Day 3: ~1 h)
    1. For data evaluation, select ten beads for each STED intensity. For each bead, extract a line (with single-pixel width) crossing the bead through its center and fit with a Gaussian function.
      Note: The fit yields a raw full width at half maximum (FWHMr), which includes the size of the dark-red beads (d0 = 40 nm). The real STED resolution (FWHM) can then be estimated as (FWHMr2 - d02)1/2. Based on the statistics of ten beads, the symbols and error bars in Figure 6A show, respectively, the means and standard deviations of the FWHMs at each STED intensity. The theoretical curve, which is fitted with the model FWHM = FWHM0/(1 + γ·ISTED2)1/2, describes the image resolution as a function of STED power. Here, FWHM0 is the resolution of the confocal microscope (with depletion beam off), ISTED is the intensity of the depletion beam, and γ is a coefficient that depends on the slope of the STED doughnut and the saturation intensity of the depletion beam (Harke et al., 2008).
    2. Compare cross-sectional intensity distributions obtained by confocal (zero depletion power) and STED imaging (150 mW depletion power) of a 40 nm bead (Figure 6B). [Example Figure 6A: FWHMs of Gaussian fits (solid lines in Figure 6A) yield FWHMr = 339 ± 11 nm for confocal mode and 50 ± 3 nm for STED mode. Removing the size of the dark-red beads, we obtain an estimated resolution of our STED microscope as 32 ± 5 nm.]


      Figure 6. Determination of the STED resolution. A. Solid squares: Quantification of the widths of cross-sections through individual beads as a function of the power of the depletion laser (see Figure 5B). Data were corrected for the intrinsic size of the beads and are shown as means ± standard deviations from ten bead images. Red line: fit with a square-root function according to STED theory. B. Cross sections through a single bead imaged at 0 and 150 mW depletion laser power (see Figure 5) showing the enormous resolution enhancement offered by STED microscopy. Gaussian fits are shown as solid lines.

  9. Imaging GFP-SiRA5 mRNA in live E. coli with STED microscopy (Day 3: ~1-2 h)
    1. Attach the temperature-controlled sample chamber to the microscope, prewarm to 37 °C and add the 8-well imaging slide used in Step E2.
    2. Set the power of the excitation and depletion beams (Example Figure 7: Excitation 5 μW, depletion 150 mW). Scan repeatedly a square region of 10 x 10 μm2, covering a group of E. coli cells, for five frames of 1,024 x 1,024 pixels (Pixel dwell time in Figure 7A: 30 μs, the main image of Figure 7A corresponds to the inset with t = 126 s).
    3. After taking the STED images, switch off the depletion laser and take a confocal image (Figure 7B).
      Note: No deconvolution was carried out on either the STED or the confocal images. Different color maps can be used for the STED and confocal images to optimally display cell structures.


      Figure 7. STED imaging of live E. coli bacteria expressing GFP-SiRA 5 mRNA. A. Time series of STED images taken in 31.5 s intervals for a total of 126 s; top: magnified image at 126 s; B. Regular confocal image taken directly after the STED time series.

Data analysis

  1. To determinate the STED resolution, statistics with multiple beads are necessary. In our experiment, ten individual beads for each STED intensity were selected to calculate the raw full width at half maximum (FWHMr) with the cross-section intensity distribution of the bead. The data points and error bars in Figure 6A present the means and standard deviations of the ten beads.
    Note: To avoid selection of a cluster of beads (rather than single DR beads), those were excluded (and replaced with new selections) when their cross-section intensity distribution has the fitted Gaussian amplitude 1.5 times higher than the averaged amplitude.
  2. To reduce the influence of sample defocusing on STED resolution determination, for each STED intensity two images were taken (from a new sample area), and the in-focus position of the sample was checked each time, which provides the maximal brightness for the beads in the confocal mode. Generally, five beads from each image were selected for the statistics of the STED resolution. However, for the case that one image has systematically-larger FWHMs than the other image (due to defocusing), ten beads from the in-focus image solely will be selected for the FWHM analysis.

Recipes

  1. M9 imaging solution (50 ml)
    1. Preparation of the 5x M9 salt broth:
      1. Add 26.25 g of M9 broth powder [Na2HPO4 (15 g), KH2PO4 (7.5 g), NaCl (1.25 g), NH4Cl (2.5 g)] into a 1,000 ml PYREX® glass bottle
      2.  Add 500 ml of MilliQ water, shake thoroughly and check the pH (7.4 at 37 °C)
      3. Autoclave with a standard liquid program
      Note: This 5x M9 mix can be stored at RT.
    2. Fill 10 ml of the 5x M9 into a 50 ml Falcon and supplement the solution with 250 μl of MgSO4 (1 M stock, 5 mM final concentration), 5 μl of CaCl2 (1 M stock, 100 μM final concentration) and 500 μl of Glucose (40% stock, final concentration 0.4%)
    3. Fill the Falcon to 50 ml with MilliQ water and filter-sterilize the solution before using it for the imaging experiment
    Note: Alternatively, the imaging experiment can also be carried out in live-cell imaging solution purchased from Thermo Fisher Scientific, supplemented with additional magnesium and glucose as described above.

Acknowledgments

M.S. and A.J. were supported by the Deutsche Forschungsgemeinschaft (Grant # Ja794/11-1), and G.U.N. by the Helmholtz Association (Program Science and Technology of Nanosystems) and the Karlsruhe School of Optics and Photonics (KSOP). This protocol was adopted from our previously published work (Wirth et al., 2019).

Competing interests

The authors declare no competing financial interest.

References

  1. Alexander, S. C. and Devaraj, N. K. (2017). Developing a fluorescent toolbox to shed light on the mysteries of RNA. Biochemistry 56(39): 5185-5193.
  2. Arora, A., Sunbul, M. and Jäschke, A. (2015). Dual-colour imaging of RNAs using quencher- and fluorophore-binding aptamers. Nucleic Acids Res 43(21): e144.
  3. 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.
  4. Bouhedda, F., Autour, A. and Ryckelynck, M. (2017). Light-up RNA aptamers and their cognate fluorogens: from their development to their applications. Int J Mol Sci 19(1).
  5. Braselmann, E., Wierzba, A. J., Polaski, J. T., Chromiński, M., Holmes, Z. E., Hung, S. T., Batan, D., Wheeler, J. R., Parker, R., Jimenez, R., Gryko, D., Batey, R. T. and Palmer, A. E. (2018). A multicolor riboswitch-based platform for imaging of RNA in live mammalian cells. Nat Chem Biol 14(10): 964-971.
  6. 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.
  7. Filonov, G. S., Moon, J. D., Svensen, N. and Jaffrey, S. R. (2014). Broccoli: rapid selection of an RNA mimic of green fluorescent protein by fluorescence-based selection and directed evolution. J Am Chem Soc 136(46): 16299-16308.
  8. Gao, P., Prunsche, B., Zhou, L., Nienhaus, K. and Nienhaus, G. U. (2017). Background suppression in fluorescence nanoscopy with stimulated emission double depletion. Nature Photonics 11: 163-169.
  9. Harke, B., Keller, J., Ullal, C. K., Westphal, V., Schonle, A. and Hell, S. W. (2008). Resolution scaling in STED microscopy. Opt Express 16(6): 4154-4162.
  10. Holeman, L. A., Robinson, S. L., Szostak, J. W. and Wilson, C. (1998). Isolation and characterization of fluorophore-binding RNA aptamers. Fold Des 3(6): 423-431.
  11. Lukinavičius, G., Umezawa, K., Olivier, N., Honigmann, A., Yang, G., Plass, T., Mueller, V., Reymond, L., Corrêa, I. R., Jr., Luo, Z. G., Schultz, C., Lemke, E. A., Heppenstall, P., Eggeling, C., Manley, S. and Johnsson, K. (2013). A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins. Nat Chem 5(2): 132-139.
  12. Murata, A., Sato, S., Kawazoe, Y. and Uesugi, M. (2011). Small-molecule fluorescent probes for specific RNA targets. Chem Commun (Camb) 47(16): 4712-4714.
  13. Neubacher, S. and Hennig, S. (2019). RNA structure and cellular applications of fluorescent light-up aptamers. Angew Chem Int Ed Engl 58(5): 1266-1279.
  14. Nienhaus, K. and Nienhaus, G. U. (2016). Where do we stand with super-resolution optical microscopy? J Mol Biol 428(2 Pt A): 308-322.
  15. Ouellet, J. (2016). RNA fluorescence with light-up aptamers. Front Chem 4: 29.
  16. Paige, J. S., Wu, K. Y. and Jaffrey, S. R. (2011). RNA mimics of green fluorescent protein. Science 333(6042): 642-646.
  17. Reshes, G., Vanounou, S., Fishov, I. and Feingold, M. (2008). Cell shape dynamics in Escherichia coli. Biophys J 94(1): 251-264.
  18. Song, W., Filonov, G. S., Kim, H., Hirsch, M., Li, X., Moon, J. D. and Jaffrey, S. R. (2017). Imaging RNA polymerase III transcription using a photostable RNA-fluorophore complex. Nat Chem Biol 13(11): 1187-1194.
  19. Sunbul, M. and Jäschke, A. (2013). Contact-mediated quenching for RNA imaging in bacteria with a fluorophore-binding aptamer. Angew Chem Int Ed Engl 52(50): 13401-13404.
  20. Sunbul, M. and Jäschke, A. (2018). SRB-2: a promiscuous rainbow aptamer for live-cell RNA imaging. Nucleic Acids Res 46(18): e110.
  21. Sunbul, M., Arora, A. and Jäschke, A. (2018). Visualizing RNA in live bacterial cells using fluorophore- and quencher-binding aptamers. Methods Mol Biol 1649: 289-304.
  22. Tan, X., Constantin, T. P., Sloane, K. L., Waggoner, A. S., Bruchez, M. P. and Armitage, B. A. (2017). Fluoromodules consisting of a promiscuous RNA aptamer and red or blue fluorogenic cyanine dyes: selection, characterization, and bioimaging. J Am Chem Soc 139(26): 9001-9009.
  23. Tyagi, S. (2009). Imaging intracellular RNA distribution and dynamics in living cells. Nat Methods 6(5): 331-338.
  24. Wang, L., Frei, M. S., Salim, A. and Johnsson, K. (2019). Small-molecule fluorescent probes for live-cell super-resolution microscopy. J Am Chem Soc 141(7): 2770-2781.
  25. Wirth, R., Gao, P., Nienhaus, G. U., Sunbul, M. and Jäschke, A. (2019). SiRA: a silicon rhodamine-binding aptamer for live-cell super-resolution RNA imaging. J Am Chem Soc 141(18): 7562-7571.
  26. Xia, Y., Zhang, R., Wang, Z., Tian, J. and Chen, X. (2017). Recent advances in high-performance fluorescent and bioluminescent RNA imaging probes. Chem Soc Rev 46(10): 2824-2843.
  27. Yerramilli, V. S. and Kim, K. H. (2018). Labeling RNAs in live cells using malachite green aptamer scaffolds as fluorescent probes. ACS Synth Biol 7(3): 758-766.

简介

[摘要 ] 遗传编码的点亮适体是显示活细胞中RNA的有前途的工具,可帮助我们加深对RNA广泛而复杂的生命的理解。可见光波长区已经被开发,他们都没有然而,据报道,在兼容先进的超分辨率技术,主要是由于不良的光物理性质其小分子荧光团。在这里,我们描述了一个详细的协议对于荧光显微镜mRNA的使用最近报道的具有优异光物理性质的荧光罗丹明结合适体(SiRA )在活细菌中进行检测。值得注意的是,我们利用SiRA 展示了首个使用超分辨率(STED)显微镜进行的基于适体的RNA可视化。这种成像方法可能特别有价值用于可视化原核生物中的RNA,因为细菌的大小仅比光学分辨率大几倍 传统显微镜的分辨率。

[背景 ] 可视化的具体RNA分子通过荧光显微镜具有不可估量的价值在过去二十年中扩大我们的知识RNA功能内的细胞在时空精气神(特亚吉,2009年;夏等人,2017年),由于缺乏。固有的荧光RNA,用于活细胞成像的荧光RNA标记工具的开发以及它们对最新显微镜的适应性 –特别是对于超分辨率显微镜– 势在必行。超分辨率显微镜(SRM)对于原核系统中的RNA成像特别有吸引力,因为细菌很小(〜2.5MYU中号长,0.5-1〜MYU 中号宽)和分辨率的标准荧光显微镜被限制在200〜300〜牛米,由于衍射极限光(Reshes 等,2008;)Nienhaus而Nienhaus年,2016年。尽管已经开发了多种工具来荧光标记感兴趣的RNA(ROI),但使用SRM(分辨率约20-50 nm)对其活细胞进行可视化仍然具有挑战性(Alexander和Devaraj,2017年),主要是因为严格对活细胞SRM所需的荧光团的光物理特性的要求,这些特性包括高光稳定性和亮度,细胞膜通透性,水溶性,吸收和发射波长,最好在远红外和近红外(NIR)区域,开关能力和成像期间足够的信噪比(Wang 等人,2019)。因此,迫切需要与SRM兼容的新型RNA报告系统。

可视化活细胞中RNA的一种特别吸引人的方法是使用基因编码的荧光发光适体,所述荧光适体是短的寡核苷酸,它们以高亲和力结合有条件的小型荧光探针,并通过指数富集(SELEX)使用配体的系统进化来开发。(Holeman et al。,1998; Ouellet,2016)。由于在小分子结合后荧光增加,因此可将aptapter标记为ROI(图1)。跨越光谱可见区的几个荧光增强RNA aptapter具有最近开发并证明其是用于RNA标记的强大工具(Bouhedda 等,2017; Neubacher和Hennig,2019)。

著名的菠菜,西兰花,芒果,玉米的适体及其改良的变体在溶液中以较低的荧光量子产率与荧光团结合,由于适体结合后分子内运动的限制而变成荧光(图1A)(Paige 等人,2011; Filonov 等人,2014; Dolgosheina 等人,2014; Song 等人,2017)。其他突出的例子是SRB-2,DNB,BHQ和Riboglow 适体,它们在结合染料或染料后会破坏淬灭染料偶联物。淬灭剂部分,从而产生荧光增强(图1B) (村田等人,2011; Sunbul而Jaschke,201 3 和2018 ; Arora的。等人,2015; Braselmann 。等人,2018)大多数这些适体-配体对。但是,MG,Riboglow ,Mango TO-3和最近报道的DI​​R-pro(尚未报道RNA标记)在远红外很受激发。 ,其荧光团显示出低的光稳定性和亮度,光毒性或 它们几乎不具有细胞渗透性(Tan 等人,2017; Autour 等人,2018; Yerramilli和Kim,2018)。

提示您通过这些缺点,我们最近开发了一种新型光了RNA适体结合3-羧基硅罗丹明(SIR )(维尔特等,2019) 。SIRS 是一个令人兴奋的新荧光基团级在现场蛋白质标记为SRM中因为他们是耐光,近红外发光的荧光团改变其平衡之间的非彩色螺内酯和荧光两性离子响应他们的环境(图1C)(Lukinavi ç 尤斯等,2013) 。这个属性是负责其高细胞通透性和荧光行为,并在过去十年中促进了SiR 染料及其衍生物的普及(Wang 等人,2019)。将SELEX应用于约2 x 10 15 的组合RNA文库中,我们发现了50 NT -Minimal适体,SIRA ,结合的目标SIR 1 用K d 430 NM 并显示出显着的荧光开启在染料结合(图2)。这一发现推出一个全新的概念Fluorogenicity 到外地适配子 的RNA成像,即环境依赖性分子内螺环化(图1C)。SiRA 显着耐受光漂白并限制迄今已知的最亮的远红光适体系统(量子产率0.98;消光系数86,000 M -1 ·c m -1 )。使用SiRA 系统,我们在免洗活细胞成像实验中可视化了细菌中RNA的表达(Wirth 等人,2019)。该协议的创新之处在于我们可以利用SiRA的出色光物理特性迄今为止,SiRA 是唯一被证明可在SRM应用中使用的发光适体系统,可用于活细菌中mRNA的刺激发射耗竭(STED)SRM 。



D:\重新格式化\ 2020-2-7 \ 1902664--1308 Murat Sunbul 791973 \无花果jpg \图1.jpg

图1. 用于RNA成像的发光RNA适体的荧光概念。A 。由于分子内运动(ON)的限制,由于溶液中分子运动(OFF)而荧光量子产率低的荧光团在适体结合后变成高荧光B.与荧光团或淬灭剂部分(ON)结合时,接触淬灭的荧光团-猝灭剂共轭物(OFF)亮起,这里示出了使用荧光团结合适体的点亮系统的例子。在溶液中,SiR 可以偏向非彩色螺内酯(OFF)。适体结合后环境的变化导致平衡向荧光两性离子(ON)的转移。



D:\重新格式化\ 2020-2-7 \ 1902664--1308 Murat Sunbul 791973 \图jpg \图2.jpg

图2. SiRA 适体及其特性A. 50 nt SiRA 适体的预测二级结构在选择过程中演变(重要特征用颜色标记)B. 该方案中使用的荧光染料1的结构,结合这SIRA 随着纳摩尔亲和力。C.标准化的吸收和发射光谱的自由爵士1 和[ SIRA * 1 ]情结。



在以下协议中,我们详细描述了用于GFP-mRNA的活细胞共聚焦和STED成像的大肠杆菌细菌的制备。我们在本文中使用基于质粒的表达系统,并用新型的串联重复序列标记GFP-mRNA。位于3 ' 非翻译区(UTR)的SiRA 适体(SiRA 5 )。该方案可适用于可视化任何质粒表达的靶RNA;但是,请记住RNA 的表达水平和半衰期。特定应用可能需要该协议的协议。

关键字:Light-up适配体, 硅-罗丹明, RNA成像, 受激发射损耗荧光显微镜, 荧光共聚焦显微镜, 活细胞成像, SiRA适配体

材料和试剂


 


移液器技巧(Sarsted t ,目录号: 70.1130,70.760.002,70.762)
1.5 ml 微量离心管(Sartsted t ,目录号:72.690.001)
15 亩-slide 8 - 良好无涂层,玻璃底部#1.5聚合物盖玻片(Ibidi GmbH公司目录编号:80827)
高Ç Larity PP Ç Onical Ç Entrifuge 吨UBE15毫升中(Falcon ® ,目录号的:352096)
高Ç Larity PP Ç Onical Ç Entrifuge 吨UBE50毫升中(Falcon ® ,目录号的:352070)
医疗的Millex -GS 小号Yringe ˚F ILTER ü NIT,0.22 亩中号(EDM默克,目录号的:SLGSM33SS)
BD Discardit d 一次性注射器10毫升(Becton Dickinson,目录号:300296)
PYREX ® 媒体乙Ottles,摹Raduated,康宁® 1000毫升(VWR ,PYREX ® ,目录号:1395-10L )
FluoSpheres TM 羧化物修饰的微球体,0.04 亩中号,暗红色荧光(六百八十○分之六百六十○),5%固体,叠氮化物不含(热飞世尔科技,产品目录号的:F8789)
金珠(GC80,BB International,目录号:EM.GC80 )
大肠杆菌BL21 Star TM (DE3)符合Invitrogen要求的大肠杆菌菌株(Thermo Fisher,目录号:C601003),存储在-80°C
pET -GFP表达质粒(pET His6 GFP TEV LIC克隆载体,1 GFP)(Addgene ,质粒编号:29663),储存在-20°C
pET-GFP-SiRA 5 表达质粒,储存在-20°C
该协议中使用的pET-GFP-SiRA 5 质粒无法商业购买,选择适体的一般串联重复序列(取决于目标RNA和表达水平)可以直接与ROI融合(在这种情况下为GFP mRNA )用适当的限制性位点的3 ' 端非编码区(在此:SalI位和XhoI位快速文摘限制酶,热科学TM ,目录号:FD0644而且FD0695,分别)如先前所描述在文献中(Sunbul 。等人,2018) 。在在这项研究中,我们根据图3 插入了5个S iRA 适体的重复序列,并带有散布的接头核苷酸。


 


D:\重新格式化\ 2020-2-7 \ 1902664--1308 Murat Sunbul 791973 \图jpg \图3.jpg


3.图摘录的PET-GFP-SIRA 5 细菌表达质粒用于GFP MRNA在这个协议中的可视化。LACO :lac操纵,RBS :核糖体结合位点,ROI:RNA的兴趣,P:启动子,T:终结者,SalI 和XhoI :限制位点,终止:终止用于RNA翻译的密码子。


 


以下序列显示了克隆表达载体(pET-GFP-SiRA 5 )以及本研究中使用的pET -GFP的摘录。


 


pET-GFP-SiRA 5 :


ShijieijijieieieishishitijitieishititishishieieitishishieiATATTGGAAGTGGATAACGGATCCG ... AATTCG AGCGCC GTCGAC AGGC CCA ShishijijijitititijieieieieishiCTGGCTGCTTCGGCAGTTGTATCCT 牛逼TGG GCCTAAGAGAGA CCA ShishijijijitititijieieieieishishiTGGCTGCTTCGGCAGTTGTATCCTT TGG TCTCAATAACCAG CCA ShishijijijitititijieieieieishiCTGGCTGCTTCGGCAGTTGTATCCT 牛逼TGG GCCTAAGAGAGA CCA ShishijijijitititijieieieieishishiTGGCTGCTTCGGCAGTTGTATCCTT TGG TCTCAATAACCAG ShishieishishijijijitititijieieieieishishiTGGCTGCTTCGGCAGTTGTATCCTT TGG CTGG CTCGAG ShieishishieishishieishishieishishieishishieishitijieijieitishishijijishitijishitieieishieieieijishishishijieieieijijieieijishitijieijititijijishitijishitijishishieishishijishitijieijishieieitieieishitieijishieitieieishishishishititijijijijishishitishitieieieishijijijitishititijieijijijijititititititijishitijieieieijijieijijieieishitieitieitishishijijieititijijishijieieitijijijieishijishijishishishitijitiAGCGGCGCATTAAGCGCGGCGGGTG ...


pET -GFP:


ShijieijijieieieishishitijitieishititishishieieitishishieiATATTGGAAGTGGATAACGGATCCG ... AATTCG AGCGCC GTCGAC AAGCTTGCGGCCGCA CTCGAG ShieishishieishishieishishieishishieishishieishitijieijieitishishijijishitijishitieieishieieieijishishishijieieieijijieieijishitijieijititijijishitijishitijishishieishishijishitijieijishieieitieieishitieijishieitieieishishishishititijijijijishishitishitieieieishijijijitishititijieijijijijititititititijishitijieieieijijieijijieieishitieitieitishishijijieititijijishijieieitijijijieishijishijishishishitijitiAGCGGCGCATTAAGCGCGGCGGGTG ...


黑色SiRA (3-nt茎环)             


灰色可变茎环或间隔区             


蓝色SalI 限制位点             


粉色XhoI 限制位点             


 


LB培养基蒸压,伦诺克斯(卡尔·罗斯®,目录号:X964.4)
LB带琼脂的肉汤,伦诺克斯(Sigma-Aldrich,目录号:L2897)
卡那链霉菌(Sigma-Aldrich,目录号:K1377)中的硫酸卡那霉素,制备1 M水溶液,过滤灭菌并在-20°C储存
-β-异丙d - 硫代- 吡喃半乳糖苷(IPTG)(Sigma-Aldrich公司,目录号的:I5502),所述的制备1M的常规水溶液,过滤灭菌,并储存于-20℃下
氧化钾颗粒EMPLURA钾® (Sigma-Aldrich公司,目录编号:1050121000),准备10 2M水溶液
聚D- 赖氨酸氢溴酸盐(Sigma-Aldrich,目录号:P7886),在DPBS中制备2.5 mg / ml,储存在4°C
Dulbecco磷酸盐缓冲盐水(DPBS)(Sigma-Aldrich,目录号:D8537)
MilliQ 水
罗丹明硅发荧光染料,100 MYU 中号方案在DMSO
该方案中使用的荧光染料不可商购,因此必须按照文献(Wirth et al。,2019)中所述通过HPLC进行合成和纯化。


注意:HPLC纯化必须非常小心,因为残留杂质的荧光会降低成像实验中的信噪比。


M9汤(Fluka Sigma-Aldrich,目录号:63011)
一水合硫酸镁(Sigma-Aldrich,目录号:434183),制备1 M水溶液并高压灭菌
D- (+)- 葡萄糖(Sigma-Aldrich,目录号:G8270),制备40%(w / v)水溶液并过滤除菌
六水合氯化镁(Sigma-Aldrich,目录号M2670),准备1 M水溶液并高压灭菌
二水合氯化钙(Sigma-Aldrich:目录号:1725801000),制备1 M水溶液并高压灭菌
活细胞成像解决方案(Thermo Fisher Scientific,目录号:A14291DJ)
M9成像解决方案(请参阅食谱)
 


配套设备


 


RES EARCH ® 加移液器,可变容积200和1000 MYU 大号(Sigma-Aldrich公司,的Eppendorf ® ,目录号小号:Z6 83817和Z683825)
蒸汽灭菌器(例如,Varioklav ® ,型号:155S)
舒适度的24 X Thermomixer中的1.5ml(的Eppendorf ® )
孵化器和振荡器(Thermo Fisher Scientific,496型)
BioPhotometer生物分光光度计(的Eppendorf ® ,6131型)
高速微量离心机(Neuation Technology Pvt。Ltd.,目录号:iFuge M12)
冷柜
电冰箱
共聚焦显微镜(尼康,型号:A1R)
在对于这个协议中,扫描共聚焦显微镜具有混合扫描器(一个点的Galvano /谐振)中的溶液中所使用的,搭载了尼康Ñ的Apo 60x的NA 1.4 的λ 小号OI(WD0.14毫米,FOV 0.21 X 0.21 MM)的目标。在对于荧光激发,488使用绿色(nm通道)和640 nm(红色通道)激光器。为了进行检测,将高灵敏度的GaAsP 检测器与525/50 nm和700/75 nm(中心波长/带宽)发射滤光片组合在一起,用于绿色和红色通道,分别使用。


STED显微镜
在STED显微镜使用这个协议是自制的,并已进行了详细描述(皋等,2017) 。此外,650纳米长通分色镜(ET700SP;色度,波纹管瀑布,VT)和需要时间相关的单光子计数(TCSPC)卡(SPC-150 ,德国柏林Becker&Hickl GmbH)。


注意:方案中的实验是在640 nm激发和779 nm耗尽的条件下进行的;仅使用了用于普通2D STED成像的涡流相板。


 


软体类


 


使用Java 1.6.0_24的ImageJ Fiji版本2.0.0。
共聚焦显微镜软件:NIS-Elements图像采集和分析软件(尼康)
STED显微镜软件:我Mspector (马普创新GmhH ,慕尼黑,德国)
Matlab(MathWorks,马萨诸塞州内蒂克)
OriginPro 2015 32位(OriginLab,Northampton,MA)
 


程序


 


大肠杆菌转化(第1天:〜2.5小时,然后过夜孵育)
注意:我们建议在每次成像实验前进行新鲜转化,以确保mRNA-SiRA 正确表达。


将50 ng 质粒(pET -GFP 和pET-GFP-SiRA 5 )加入每个高压灭菌的微量离心管中,并置于冰上。
在冰上融化化学感受态大肠杆菌BL21 Star TM (DE3)细胞15分钟。
40添加MYU 大号中大肠杆菌BL21 星TM (DE3)细胞到每个管中并孵育另外30分钟冰上。
将试管在42°C下加热45 s,然后在冰上孵育2分钟。
250添加Myueru 预温热(37℃)的LB培养基中向每管摇晃在650rpm下60分钟。
路径选择的细菌携带质粒,条纹20 MYU 大号的细菌在LB琼脂平板含有30中MYU ģ /毫升卡那霉素。
将LB琼脂平板在37 °C下孵育过夜。
注意:早晨,将琼脂平板置于4°C,并整天放置在那里。


 


诱导细菌中的mRNA表达(第2天:15分钟,然后进行过夜孵育;第3天:〜5 小时)
加入5毫升LB培养基中添加30 MYU 摹/ ml卡那霉素进入15毫升˚F 爱尔康并启动过夜培养使用一个单一的殖民地采摘从每个LB琼脂平板上使用无菌枪头。
所述孵育˚F 爱尔康在37℃下剧烈振荡以170rpm过夜。
第二天早晨,测量每个过夜培养物以1:10稀释的光密度(OD 600 )。
起始培养物产生来自过夜培养。对于这一点,添加10ml的LB培养基中添加30 MYU ģ / ml卡那霉素在50毫升˚F 爱尔康并添加过夜培养,得到OD 600 为0.05。
在37°C和170 rpm摇动培养物,直到OD 600 达到0.35。
注意:这通常需要1.5-2小时。


通过加入10中诱导GFP MRNA的表达MYU 大号中IPTG(终浓度为1mM)并摇动培养另外3小时,在37℃下
注意:目前SiRA 系统仅用于高表达的GFP mRNA的成像。为了可视化较低的拷贝数,可以设计更多的串联重复序列,并且可能需要进一步优化方案(例如染料浓度)。


 


制备聚D- 赖氨酸涂层的玻璃室载玻片(第3天:〜3小时,与步骤B 5- B 6 平行)
注意:用于涂层的聚-D-赖氨酸溶液需要在每次将其添加到孔中之前新鲜制备。但是,聚-D-赖氨酸涂层的玻璃室载玻片可以提前24小时制备并保存放入6 °C 的冰箱中。建议在使用前将其加热至室温。


删除8孔Ibidi 玻片从包装并孵育每孔200 MYU 大号的10 1M KOH水溶液5分钟。
KOH溶液取下井很细心,把它们洗干净彻底多次使用400 MYU 大号无菌的MilliQ 水和空气晾干60分钟。
1000混合MYU 大号水与20 MYU 大号的聚- d -赖氨酸库存(〜50 MYU ģ / MYU 大号终浓度),并加入250 MYU 大号的溶液,快速加入每个孔中(食谱4个孔)。
在室温下孵育孔45分钟。
每口井洗两次用300 MYU 大号无菌的MilliQ 水和空气干燥至少30分钟前的固定化细菌。
 


制备大肠杆菌样品进行成像(第3天:〜1小时)
注意:对于活细胞成像实验,由于短的mRNA半衰期和细菌代谢,我们建议一次最多成像2个孔。


将每种培养物的200μl 等分试样(步骤B6)转移到高压灭菌的微量离心管中,并在室温下以5,600 x g 沉淀细胞2分钟。
除去LB培养基,并将细菌悬浮在1 ml M9成像溶液中(配方1)。
200添加MYU 大号细胞悬浮到每个孔中的经涂覆的8孔Ibidi 幻灯片。
在室温下孵育细菌35分钟,以使其粘附在表面上。
每个孔的洗涤轻轻地用400 Myueru 中M9成像解决方案。
300添加Myueru 中M9的成像解决方案包含500 NM 的硅罗丹明发荧光染料,直接准备用于映像。
注意:染料在37°C时会很快渗透到细菌中(5-10分钟);因此,显微镜调整的时间足以进行孵育。如果出现渗透问题,则将染料/ M9成像溶液预热至37建议使用℃ 。


 


使用共聚焦显微镜对GFP-SiRA 5 mRNA 成像(第3天:〜1-2 小时)
打开488 nm(绿色通道)和640 nm(红色通道)激光以进行点扫描共聚焦显微镜的荧光照明,并分别将525/50 nm和700/75 nm发射滤光片用于绿色和红色通道。
将温度培养箱连接至显微镜,预热至37°C,然后将8孔载玻片和附着的大肠杆菌放入培养箱中。
亮使用场照明集中于被固定的大肠杆菌与尼康Ñ的Apo 60X NA 1.4 Ramuda 小号OI(WD为0.14mm,FOV 0.21 X 0.21毫米)目的,开始与PET-GFP-SiRA 5转化细胞。
为GFP和红色通道以及正确的z轴确定合适的曝光时间。选择设置以使像素不饱和但仍可获得最高的荧光信号。
采集所有通道的明场和荧光图像(图4)。
 


D:\重新格式化\ 2020-2-7 \ 1902664--1308 Murat Sunbul 791973 \图jpg \图4.jpg


图4. 使用共聚焦显微镜在活大肠杆菌中进行GFP mRNA成像的实例A. 成像实验中所用质粒的示意图荧光团与SiRA 适体的结合在显微镜的红色检测通道中产生荧光信号。 B. 大肠杆菌活细胞中GFP mRNA的免洗活细胞成像。用GFP质粒或GFP-SiRA 5 质粒转化细胞,并用明场,绿色(蛋白质表达)拍摄图像)和红信道(检测mRNA)。量表酒吧= 5 Myuemu。C.放大描述于图4B中的图像。量表酒吧= 5 Myuemu。


 


使用相同的显微镜设置,对带有对照质粒pET -GFP的大肠杆菌细胞重复步骤E3-E5 。
为了进行背景校正,我们使用Fiji / Image J图像分析软件,手动选择没有附着大肠杆菌细胞的表面积,并从整个图像中减去其平均荧光信号。
 


 


(可选的)调整的STED显微镜ë
通过扫描两个颜色通道中的检测到金珠(GC80),确保640 nm激发光束和779 nm耗尽光束的焦点在空间和时间上重叠。
首先,用6个50-nm长通二向色镜将反射/散射的激发和耗尽光束分成两束,然后通过780/10 nm(中心/宽度)和640的带通滤光片过滤这两束光/ 10 nm,直到被两个雪崩光电二极管(APD)检测到为止。
使用Imspector 软件同步扫描信号和光子检测间隔,从而生成没有光谱串扰的两通道图像。


注意:如有必要,需要稍微调整耗尽光束,以确保STED图像(具有类似甜甜圈轮廓的轮廓)的中心与激发图像(具有高斯轮廓)精确重叠。


检查激发和耗尽脉冲之间的时间重叠,然后使用时间相关的单光子计数(TCSPC)卡收集金珠反射的640 nm和779 nm反射光,并根据常见触发对其进行分类。可以在TCSPC软件的示波器显示模块中监控640 nm激发脉冲与779 nm脉冲之间的延迟。
注意:为了获得最佳的耗尽,可以使用一个延迟盒来调整激发脉冲和耗尽脉冲之间的延迟(在我们的示例中为200 ps左右),该延迟盒会将耗尽激光器的触发输出路由到激发激光器的触发输入。


 


成像深红色珠(第3天:〜1小时)
注意:暗红色珠子的图像用于确定STED显微镜的分辨率,应在样品制备之前或平行进行。


稀Fluosphere TM 40-NM暗红色珠(六百八分之六百六十零)从原料溶液进入PBS在体积比为1:10 4 。
200添加MYU 大号的溶液,其中的多晶硅- d -赖氨酸涂层成像房和孵育5分钟在室温下,以固定珠的表面。
盖玻片清洗两次,用250 Myueru 的MilliQ 水,最后加250 Myueru 的MilliQ 水成像。
将盖玻片放在显微镜载玻片架上。
调整激励功率向5- MYU W¯¯ 在样品和图片与耗尽激光以不同功率介于0 MW 为150 兆瓦。扫描的矩形区域10的X 10 MYU 中号2 带1 ,024 X 1 ,024像素,覆盖数十单个深红色珠子。
注意:为最大程度减少在同一区域进行多次扫描时的光漂白现象,建议在不同的STED激光强度下使用新鲜区域进行STED成像。


在共焦和STED模式之间生成可比较的图像(图5A)。在那里,通过共焦和STED显微镜对相同区域依次成像。
注意:通过改变耗尽功率,可以改变有效分辨率,如在耗尽束的不同功率下拍摄的珠子图像所示(图5B)。


 


D:\重新格式化\ 2020-2-7 \ 1902664--1308 Murat Sunbul 791973 \无花果jpg \图5.jpg


5.图暗红色的珠子图像A. 共聚焦(上左)和STED(右下)图像的荧光珠在玻璃盖玻片,显示出巨大的决议ENH Ancement由于STED成像。规模酒吧= 3 Myuemu 。B. 以耗尽束的不同功率拍摄的单个珠子的图像。


 


使用深红色珠子图像评估STED分辨率(第3天:〜1小时)
为了进行数据评估,为每个STED强度选择十个珠子。对于每个珠子,提取一条穿过珠子中心的线(具有单个像素宽度),并与高斯函数拟合。
注意:拟合产生的原始半峰全宽(FWHM r )包括深红色珠子的尺寸(d 0 = 40 nm),然后可以将实际STED分辨率(FWHM)估算为(FWHM r 2 - d 0 2 )1/2 。据统计10个珠,符号和误差条来图6A显示,分别为平均值和标准偏差半值宽度在每个STED强度理论曲线,其装配中。示范FWHM随着= FWHM 0 /(1 Tasu 赶马 我STED 2 )1/2 ,描述了图像分辨率作为功能的STED力。在此,FWHM 0 是分辨率的共聚焦显微镜(枯竭梁关), I STED 是耗尽束的强度,而γ 是取决于STED甜甜圈的斜率和耗尽束的饱和强度的系数(Harke等,2008)。


比较通过共聚焦(零耗尽功率)和40 nm磁珠的STED成像(150 mW 耗尽功率)获得的横截面强度分布(图6B)(示例图6A:高斯拟合的半峰宽(图6A中的实线)收率FWHM - [R = 339±11nm的共聚焦模式和50±3 Nm,用于STED模式。卸下暗红色小珠的大小,我们获得估计的拆分我们STED显微镜32±5nm以下。]
 


D:\重新格式化\ 2020-2-7 \ 1902664--1308 Murat Sunbul 791973 \无花果jpg \图6 .jpg


图6. STED分辨率的确定A. 实心正方形:通过单个磁珠的横截面宽度的量化与耗尽激光功率的函数关系(见图5B)。对数据的固有尺寸进行了校正。红线:根据STED 理论与平方根函数拟合B. 在0和150 mW 耗尽激光功率下成像的单个磁珠的横截面(见图5) )显示了STED显微镜所提供的巨大分辨率增强效果,高斯拟合以实线显示。


 


用STED显微镜对活大肠杆菌中的GFP -SiRA 5 mRNA进行成像(第3天:〜1-2 小时)
将温度受控的样品室连接到显微镜,预热至37°C,并添加在步骤E2中使用的8孔成像玻片。
功率设定激发和耗竭梁(实施例图7:励磁5 MYU w ^ ,折耗150 MW 。)扫描反复的正方形区域为10×10 MYU 中号2 ,覆盖一组大肠杆菌细胞,五帧1 ,024 X 1 ,024象素(象素驻留时间。在图7A:30 MYU 小号,主图像图7A对应于插图为t = 126 S)。
拍摄STED图像后,关闭耗尽激光并拍摄共聚焦图像(图7B)。
注意:在STED或共聚焦图像上均未进行反卷积,可将不同的颜色图用于STED和共聚焦图像以最佳地显示细胞结构。


 


D:\重新格式化\ 2020-2-7 \ 1902664--1308 Murat Sunbul 791973 \图jpg \图7.jpg


7. STED成像图活大肠杆菌细菌中表达GFP-SIRA 5 mRNA的表达。A.时间序列拍摄于31.5秒的间隔,总共126 S的STED图像;顶部:放大图像在126 S; B.正常共焦成像直接在STED时间序列之后拍摄。


 


资料分析


 


为了确定STED分辨率,需要使用多个珠子进行统计。在我们的实验中,针对每个STED强度选择十个单个珠子,以计算珠子的横截面强度分布来计算半峰全宽(FWHMr )。图6A中的数据点和误差线显示了十个磁珠的平均值和标准偏差。
注意:为避免选择一串珠子(而不是单个DR珠子),当它们的横截面强度分布的拟合高斯振幅比平均振幅高1.5倍时,将其排除(并替换为新的选择)。


为了减少样品散焦对STED分辨率测定的影响,对于每个STED强度,均拍摄了两张图像(来自新的样品区域),并且每次都检查了样品的对焦位置,这为微珠提供了最大的亮度通常,从每个图像中选择五个小珠进行STED分辨率统计,但是,对于一个图像具有系统地大于另一个图像的FWHM(由于散焦)的情况,则从聚焦图像将仅用于FWHM分析。
 


菜谱


 


M9成像溶液(50毫升)
5x M9盐肉汤的制备:
26.25克添加M9肉汤粉末的[Na 2 HPO 4 (15克),KH 2 PO 4 (7.5克),氯化钠(1.25克),NH 4 氯(2.5克)]到1000ml PYREX ® 玻璃瓶
加入500 ml MilliQ 水,充分摇晃并检查pH值(在37°C时为7.4)
带有标准液体程序的高压灭菌器
注意:此5x M9混合物可以在RT上存储。


填充将10ml 5×M9成一个50毫升˚F 爱尔康和补充用250溶液微升的硫酸镁4 (1M的母液,5mM的终浓度),5 微升的CaCl 2 (1M,股票,100 亩中号终浓度) 500和Myueru 葡萄糖(40 Pasento股票,终浓度0.4 Pasento)
填充˚F 爱尔康到了50毫升的MilliQ 水和过滤,消毒解决方案之前,将其用于成像实验
注意:另外,成像实验也可以在购自赛默飞世尔科技(Thermo Fisher Scientific)的活细胞成像溶液中进行,并如上所述补充镁和葡萄糖。


 


致谢


 


而MS AJ 是支持由德国研究联合会(格兰特#Ja794 / 11-1)和喷枪由亥姆霍兹联合会(计划科技纳米系统)和卡尔斯鲁厄学校光学和光子学(KSOP)。该协议获得通过从我们之前发表的作品(Wirth 等,2019)。


 


Ç Ompeting兴趣


 


作者声明没有竞争性的经济利益。


 


参考文献


 


Alexander SC和DevarajNK(2017)。开发荧光工具箱阐明RNA的奥秘。生物化学56(39):5185-5193。
Arora的,A.,Sunbul ,M。和Ĵ 甲Schke ,A.(2015)。双彩色成像的RNA使用淬灭剂-荧光团和结合性的适体。核酸研究43(21):E144。
Autour ,A.,S,CYJ,A,DC,Abdolahzadeh ,A.,Galli,A.,Panchapakesan,SSS,Rueda,D.,Ryckelynck ,M.和Unrau ,PJ(2018)。用于成像的荧光RNA芒果适体。哺乳动物细胞中的小非编码 RNA。Nat Commun 9(1):656。
Bouhedda ,F.,Autour ,A.和Ryckelynck ,M.(2017年)。点亮的RNA适体及其同源氟:从其开发到应用,《国际分子生物学杂志》19(1)。
Braselmann ,E.,Wierzba ,AJ,Polaski ,JT,Chromiński ,M.,Holmes,ZE,Hung,ST,Batan ,D.Wheeler ,JR,Parker,R。,Jimenez,R.,Gryko ,D。,RT,Batey ,RT和Palmer,AE(2018)。基于多色核糖开关的平台,用于在活的哺乳动物细胞中对RNA成像 .Nat Chem Biol 14(10):964-971。
Dolgosheina ,EV,钲,SC,Panchapakesan,SS,Cojocaru ,R.,陈,PS,威尔逊,PD,霍金斯,N.,威金斯,PA和Unrau ,PJ(2014)。RNA芒果适体-荧光:一个明亮,用于RNA标记和跟踪的高亲和力复合物 .ACS Chem Biol 9(10):2412-2420。
Filonov ,GS,Moon,JD,Svensen ,N。和Jaffrey,SR(2014)。花椰菜:通过基于荧光的选择和定向进化快速选择绿色荧光蛋白的RNA模仿物,《美国化学学会杂志》136(46): 16299-16308。
ģ 鳌,P.,Prunsche,B.,周,L.,Nienhaus,K。和Nienhaus,GU(2017)。背景抑制在荧光纳米显微随着受激发射损耗双人。自然光子学11:163-169。
Harke ,B.,Keller,J.,Ullal ,CK,Westphal,V.,Schonle ,A.和Hell,SW(2008)。STED 显微镜中的分辨率缩放 .Opt Express 16(6):4154-4162。
Holeman,LA,Robinson,SL,Szostak ,JW和Wilson,C。(1998)。荧光团结合RNA适体的分离和表征, Fold Des 3(6):423-431。
Lukinavicius ,G.,梅泽,K.,奥利弗,N.,霍尼曼,A.,杨,G.,普拉斯,T.穆勒,五,雷蒙,L.,科尔ē 一,IR,小,罗,ZG,Schultz,C.,Lemke,EA,Heppenstall ,P.,Eggeling ,C.,Manley,S。和Johnsson ,K。(2013)。一种用于细胞活细胞超分辨率显微镜的近红外荧光团。 。蛋白质纳特化学5(2):132-139。
村田,A.,佐藤,S.,Kawazoe,Y。和上杉,M。(2011)。小分子荧光探针为特定的RNA靶。化学COMMUN (CAMB )47(16):4712-4714。
Neubacher 。,S。和亨尼格,S。(2019)RNA结构和细胞应用荧光光了适体。 Angew 化学诠释埃德英格兰58(5):1266至1279年。
Nienhaus,K.和Nienhaus,GU(2016)。我们在超分辨率光学显微镜下的立场是什么? J Mol Biol 428(2 Pt A):308-322。
Ouellet,J.(2016)。带发光适体的RNA荧光。前沿化学4:29 。
Paige,JS,Wu,KY和Jaffrey,SR(2011)。绿色荧光蛋白的 RNA 模仿。科学333(6042):642-646。
Reshes ,G.,Vanounou ,S.,Fishov ,I。和Feingold的,M。(2008)。细胞形状动力学在大肠杆菌。生物物理Ĵ 94(1):251-264。
Song,W.,Filonov ,GS,Kim,H.,Hirsch,M.,Li,X.,Moon,JD和Jaffrey,SR(2017)。使用光稳定的RNA-荧光团复合物成像RNA聚合酶III转录。Nat Chem生物化学13(11):1187-1194。
Sunbul ,M。和Ĵ 甲Schke ,A.(2013)。联系介导的淬火对于RNA成像在细菌用荧光团-结合适体。 Angew 化学诠释埃德英格兰52(50):13401-13404。
Sunbul ,M。和Ĵ 甲 Schke ,A。(2018)。SRB-2 :.混杂彩虹适体的活细胞成像RNA核酸研究46(18):E110。
Sunbul ,M.,Arora的,A。和Ĵ 甲Schke ,A。(2018)。可视化RNA在活细菌细胞中使用荧光团和猝灭剂,结合适体。方法分子生物学杂志1649:289-304。
Tan,X.,Constantin,TP,Sloane,KL,Waggoner,AS,Bruchez ,MP和Armitage,BA(2017)荧光模块由混杂的RNA适体和红色或蓝色的荧光花青染料组成:选择,表征和生物成像。 J Am Chem Soc 139(26):9001-9009。
Tyagi,S.(2009)。成像活细胞中细胞内RNA分布和动力学。Nat Methods 6(5):331-338。
Wang,L.,Frei,MS,Salim,A.和Johnsson ,K.(2019)。用于活细胞超分辨率显微镜的小分子荧光探针 .J Am Chem Soc 141(7):2770-2781。
维尔特,R.,高,P.,Nienhaus,GU,Sunbul,M和J 一Schke,A.(2019)。SIRA :.硅罗丹明结合性适配体活细胞超分辨率成像RNA Ĵ化学会Soc 141(18):7562-7571。
Xia,Y.,Zhang,R.,Wang,Z.,Tian,J. and Chen,X.(2017)。高性能荧光和生物发光RNA成像探针的最新进展 .Chem Soc Rev 46(10):2824 -2843。
Yerramilli ,VS和Kim,KH(2018)。使用孔雀石绿适体支架作为荧光探针标记活细胞中的RNA.ACS Synth Biol 7(3):758-766。
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引用:Wirth, R., Gao, P., Nienhaus, G. U., Sunbul, M. and Jäschke, A. (2020). Confocal and Super-resolution Imaging of RNA in Live Bacteria Using a Fluorogenic Silicon Rhodamine-binding Aptamer. Bio-protocol 10(9): e3603. DOI: 10.21769/BioProtoc.3603.
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