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Sep 2020

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Single-molecule Fluorescence Technique to Monitor the Co-transcriptional Formation of G-quadruplex and R-loop Structures
单分子荧光技术监测G-四链体和R-环结构的共转录形成   

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Abstract

G-quadruplexes (GQ) and R-loops are non-canonical nucleic acid structures related to gene regulation and genome instability that can be formed during transcription; however, their formation mechanisms remain elusive. To address this question, we developed a single-molecule fluorescence technique to monitor the formation of G-quadruplex and R-loop structures during transcription. Using this technique, we found that R-loop formation precedes GQ formation and that there exists a positive feedback loop between G-quadruplex and R-loop formation.

Keywords: Single-molecule fluorescence (单分子荧光), G-quadruplex (G-四链体), R-loop (R环), FRET (FRET), Transcription (转录)

Background

G-quadruplexes (GQ) consist of stacked G-tetrads that are formed from four Hoogsteen base-paired guanines (Gellert et al., 1962). GQ formed at certain hotspots in the genome (Biffi et al., 2013; Marsico et al., 2019) play regulatory roles (Siddiqui-Jain et al., 2002; Falabella et al., 2019) and are related to certain diseases (Biffi et al., 2014; De Magis et al., 2019). R-loops are three-stranded nucleic acid structures composed of a double-stranded DNA-RNA hybrid and a single-stranded DNA. R-loops are also related to gene regulation and genome instability (Aguilera and García-Muse 2012; Santos-Pereira and Aguilera 2015). Interestingly, GQ and R-loops are formed during transcription and often coexist (Duquette et al., 2004).


The biological roles of GQ and R-loops and their formation mechanisms have been extensively studied over the last few decades. GQ and R-loop formation has previously been detected using bisulfite sequencing (Roy and Lieber 2009) or sequencing after immunoprecipitation (Chan et al., 2014; Hansel-Hertsch et al., 2018). Fluorescent ligands that bind specifically to GQ have also been used to observe GQ formation (Kreig et al., 2015). These techniques have provided understanding of the hotspots where GQ and R-loops are often formed (Sanz et al., 2016), in addition to the enzymes that regulate the formation of GQ and R-loops (Masse and Drolet 1999); however, their exact formation mechanisms remain to be elucidated.


Here, we report single-molecule fluorescence techniques that monitor the cotranscriptional formation of GQ and R-loops. To detect GQ formation, we used FRET (Fluorescence Resonance Energy Transfer), and to detect R-loop formation, we used the fluorescently labeled S9.6 antibody (Figure 1). We monitored GQ and R-loop formation during transcription and found that R-loop formation precedes GQ formation and there exists a positive feedback loop between GQ and R-loop formation.



Figure 1. Single-molecule fluorescence technique to monitor the cotranscriptional formation of GQ coupled with R-loops. GQ and R-loop formation is monitored by high FRET efficiency and labeled S9.6 antibody, respectively.


Materials and Reagents

  1. Double-sided tape (3M, catalog number: 137-ROK)

  2. Polyethylene tubing (BD Intramedic, Fisher Scientific, catalog number: 427411)

  3. Quartz microscope slide (H. FINKENBEINER Inc., USA, catalog number: custom-order, size : 1’’ × 3’’ × 1mm)

  4. Micro cover glass (VWR, USA, catalog number: 48393230)

  5. DNA oligos (Figure 2)

    1. Non-template strand (1/2; purchased from IDT):

      ATCAGGTCTAATACGACTCACTATAGGAAGAGAAAGT/iCy5/TTCTGGGAGGG

    2. Non-template strand (2/2; purchased from IDT):

      /5Phos/AGGGAGGGTGTA/iCy3/CTGATGCGTTCCACTCGC

    3. Template strand (1/1; purchased from IDT):

      GCGAGTGGAACGCATCAGTACACCCTCCCTCCCTCCCAGAAACTTTCTCTTCCTATAGTGAGTCGTATTAGACCTGAT/biotin/



    Figure 2. Sample design. DNA substrate is made using annealing and ligation (top). DNA substrate contains a T7 promoter and a GQ-forming sequence. Biotin (pink) is labeled at the upstream end for surface immobilization, and Cy3 (green) and Cy5 (red) are labeled as a FRET pair to detect GQ formation.


  6. Distilled water

  7. Liquid nitrogen

  8. Acrylamide:bis=29:1 (30%) (Biosesang, catalog number: A2003-1)

  9. 10× TBE (Biosesang, catalog number: TR2004-100-00)

  10. N, N, N’, N’-tetra-methylethylenediamine (TEMED) (Bio-Rad, catalog number: 161-0801)

  11. Ammonium persulfate (APS) (Sigma-Aldrich, catalog number: 1001799334)

  12. Formamide (Sigma-Aldrich, catalog number: 01246)

  13. UREA (Bio-Rad, catalog number: 161-0731)

  14. Ethanol (Carlo Ebra Reagents, catalog number: 528167)

  15. Alexa Fluor 488 C5-maleimide (Thermo Fisher, Invitrogen, catalog number: A10254)

  16. NAP-5-column (GE Healthcare, catalog number: 17-0853-02)

  17. Primary antibody: Anti-DNA-RNA hybrid (S9.6) antibody (Kerafast, catalog number: ENH001)

    Note: We divide the stock solution of S9.6 antibody into aliquots, freeze each tube in liquid nitrogen, and store them at -20°C (short term) or -80°C (long term).

  18. Secondary antibody: Anti-Mouse IgG (H+L) (Jackson ImmunoResearch, catalog number: 715-005-151)

  19. Sulfuric acid (Daejung, catalog number: 7664-93-9)

  20. Hydrogen peroxide solution (30%) (DaeJung, catalog number: 7722-84-1)

  21. Biotin-PEG-SC (Laysan Bio Inc., catalog number: 141-63)

  22. mPEG-SVA (Laysan Bio Inc., catalog number: 149-75)

  23. Epoxy glue (LOCTITE, catalog number: 326795)

  24. T4 DNA ligase (New England Bio Labs, catalog number: M0202M)

  25. 10× T4 DNA ligase buffer (New England Bio Labs, catalog number: B0202S)

  26. T7 RNA polymerase (New England Bio Labs, catalog number: M0251S)

  27. Trolox (Sigma-Aldrich, catalog number: 238813)

  28. PCD (Oriental Yeast Co., catalog number: 46852004)

  29. PCA (Sigma-Aldrich, catalog number: 37580)

  30. Spermidine (Sgima-Aldrich, catalog number: 85558)

    Note: Only a small amount to be used within a week is diluted and stored in a drawer from which light is completely blocked. After using the stock solution, the air in the container should be replaced with nitrogen gas and stored at room temperature.

  31. rNTP set (GE Healthcare, catalog number: 27202501)

  32. 1,4-dithiothreitol (Merck, catalog number: 222-468-7)

    Note: DTT is known to be easily oxidized in water over time. To prevent degradation, divide 1 M DTT into aliquots and store them at -20°C after dissolving. Moreover, it is recommended not to use DTT solution several months after making it even if it has been stored at -20°C.

  33. Acetic acid (J.T.Baker, Fisher Scientific, catalog number: 14-650-388)

  34. Sodium bicarbonate (Fisher Scientific, catalog number: S233-500)

  35. Sodium borate (Fisher Scientific, catalog number: S248-500)

  36. Aminopropylsilane (UCT SPERCIALTIES, LLC, catalog number: A0700)

  37. Fluorescent beads (FluoSpheres, Invitrogen, catalog number: 1890851)

  38. 10× TBE buffer (Biosesang, catalog number: TR2004-100-00)

  39. PCR purification kit (QIAGEN, catalog number: 28106)

  40. Trolox solution (see Recipes)

  41. PCA solution (see Recipes)

  42. T50 buffer (see Recipes)

  43. Denaturing gel solution (see Recipes)

  44. Piranha solution (see Recipes)

  45. Silanization solution (see Recipes)

  46. PEGylation solution (see Recipes)

  47. Imaging buffer (see Recipes)

  48. 4× elongation buffer (see Recipes)

Equipment

  1. Home-built TIRF microscope setup

    Note: The components used in the home-built total internal reflection fluorescence (TIRF) microscope system are as follows: EMCCD, microscope, lasers (473-nm, 532-nm, 633-nm), shutter controller, prism, dichroic mirrors, piezo stage, and microscope temperature control system. A detailed experimental setup can be found in previously published papers (Roy et al., 2008; Lee et al., 2010).

  2. Microscope temperature control system (Live Cell Instrument, model: CU-109)

  3. Incubator (FINE PCR, model: ALB 6400)

  4. Thermal cycler (Bio-Rad, model: C1000)

  5. Vertical electrophoresis cell (Mini-PROTEAN) (Bio-Rad, model: 1658000 FC)

  6. Gel documentation system (SYNGENE, model: G:box Chemi XT4 system)

  7. Ultrasonic cleaner (used as a water bath) (Branson, model: 3510E-DTH)

    Note: The ultrasonic cleaner can serve as a water bath in heating mode. We set the temperature to 69°C, but the actual temperature was roughly 55°C when running the denaturing gel.

  8. Razor (Dorco, South Korea)

  9. Diamond solid thin drill (OD: 0.75 mm) (UKAM Industrial Superhard Tools, catalog number: 2040075)

  10. Glass jar (for Piranha)

  11. Water purification system (Millipore, model: ZRXQ-003-EU)

  12. Syringe pump (HARVARD APPARATUS, model: PHD2000)

  13. Spectrophotometer (Thermo Scientific, model: NANODROP 2000)

Software

  1. Labview (version: 2015) (National Instruments, USA, https://www.ni.com/)

  2. IDL (version: 7.0) (David Stern & ITT Visual Information Solutions, USA, https://www.l3harrisgeospatial.com/Software-Technology/IDL)

  3. Matlab (version: R2015a) (MathWorks, USA, https://www.mathworks.com)

  4. Origin (version: 8.5) (Electronic Arts, USA, https://www.origin.com)

Procedure

  1. Sample preparation

    1. DNA ligation

      1. Annealing

        1. Prepare PCR-size tubes that are compatible with the use of a thermal cycler.

        2. Mix 4 µl each 100 µM DNA oligo in T50 buffer by pipetting (final concentration: 5 µM, volume: 80 µl).

        3. Place the tube containing the oligos in a thermal cycler.

        4. Incubate the solution for 3 min at 95°C.

        5. Cool the solution slowly from 80°C to 25°C. A cooling speed of -1°C per min should be adequate.

        6. Maintain the temperature of the thermal cycler at 4°C after annealing.

      2. Ligation

        1. Transfer the annealed DNA to a 1.7-ml tube.

        2. Add 60 µl distilled water.

        3. Add 16 µl 10× T4 ligase buffer.

        4. Mix the solution by pipetting.

        5. Add 4 µl 5× T4 DNA ligase (Total volume of the solution: 160 µl).

        6. Mix the solution by pipetting and store in an incubator maintained at 16°C for 16 h.

        7. Add 80 µl 5 M NaCl and 560 µl EtOH to the 160 µl solution (after ligation) and mix by pipetting.

        8. Incubate the solution at -20°C for more than 2 h.

        9. Centrifuge the solution at 4°C (rcf: 16,100 × g, and use balancer).

        10. Remove the buffer. Be sure not to suck up the pellet.

        11. Dry the pellet by leaving the lid open inside a drawer at room temperature for 30 min to eliminate any remaining EtOH.

        12. Dissolve the pellet in 4 µl distilled water and add 4 µl 99% formamide.

      3. Purification

        1. Prepare the gel-casting chamber (1-mm thick) and wash with distilled water.

        2. Make the denaturing gel (see Recipes).

        3. Add 7 µl TEMED and 60 µl 20% APS and mix well.

          Note: When adding TEMED and APS, the denaturing gel solution should be cool enough to prevent an abrupt solidification before pouring it into the casting chamber.

        4. Pour the solution into the interstice of the gel plate set and insert a comb.

        5. Wait until the gel has completely solidified.

        6. Assemble the inner chamber and mount in the outer chamber.

        7. Make 0.5× TBE buffer and pour into the inner and outer chambers.

        8. Place the outer chamber in a heat bath maintained at 55°C.

        9. Before pre-running, wash the wells by pipetting.

        10. Run the gel at 200 V without any sample loaded for more than 30 min.

        11. Stop the pre-running and wash the wells again by pipetting.

        12. Load 8 µl sample in each well.

        13. Run the gel again for 40 min at 200 V.

        14. Stop the gel running and take a picture of the gel under a red-colored lamp.

        15. Extract only the first band at the top using a razor.

          Note: The other bands below the first band correspond to DNA fragments that are not properly ligated.

        16. Prepare the gel-purification kit and place the gel slices in a column.

        17. Pour 200 µl distilled water into the column and incubate for 16 h at room temperature.

      4. Recovery and re-annealing

        1. Centrifuge the purification kit at 16,100 × g for 1 min.

        2. Collect 180 µl purified DNA and mix with 90 µl 5 M NaCl and 630 µl EtOH.

        3. Store at -20°C for 2 h.

        4. Centrifuge at 4°C for 40 min.

        5. Remove the solution.

        6. Dry the open tube in a drawer for 30 min to eliminate any remaining EtOH.

        7. Dissolve the pellet in 10 µl distilled water and mix with 0.2 µl 1 M LiCl and 0.4 µl 1 M Tris-HCl (pH 8.0).

          Note: We used LiCl instead of NaCl to prevent G-quadruplex formation during the annealing process.

        8. Transfer to a PCR-size tube.

        9. Place the tube in a thermal cycler.

        10. Incubate for 3 min at 95°C

        11. Cool the sample slowly from 80°C to 25°C. A cooling speed of -1°C per min is sufficient.

        12. Store at -20°C.

    2. Antibody labeling

      1. Dissolve 1 mg maleimide Alexa Fluor 488 dye in 55 µl DMSO.

      2. Mix 50 µl antibody and 2 µl dye in 100 mM sodium bicarbonate solution.

      3. Incubate the mixture for 2 h at room temperature.

      4. Eliminate the unreacted dye by purification using an NAP5-column.

      5. Check the labeling efficiency using a spectrophotometer.

      6. Store the labeled antibody at 4°C.


  2. Flow cell preparation

    1. Hole making

      Make holes in a quartz slide using a diamond drill bit (Figure 3).

      Note: If the size of the holes is too large, epoxy glue can leak into the inner space of the flow cell. To prevent this, it is important to compare the size of the holes with the diameter of the PE tubing during drilling.

    2. PEG coating

      1. Cleaning

        1. Place cover glasses and quartz slides into glass jars.

        2. Rinse cover glasses and quartz slides with distilled water several times.

        3. Pour Piranha solution (see Recipes) into the jars and incubate for 20-30 min to clean the cover glasses and quartz slides.

        4. Repeat step iii another 3 times.

        5. Rinse the cover glasses and quartz slides with distilled water several times.

      2. Silanization

        1. Rinse the cleaned cover glasses and quartz slides twice with methanol.

        2. Pour silanization solution (see Recipes) into the jars.

        3. Remove the solution after a 30-min reaction.

        4. Rinse the cover glasses and quartz slides with methanol and subsequently with distilled water.

        5. Remove the cover glasses and quartz slides from the jars and dry by blowing nitrogen gas.

      3. PEGylation

        1. Make PEGylation solution (see Recipes).

        2. Drop 70 µl PEGylation solution on a quartz slide.

        3. Place a cover glass on top.

        4. Incubate in a drawer for 4 h.

        5. Store at -20°C after incubation.

    3. Flow cell assembly (Figure 3)

      1. Thaw and rinse the PEGylated cover glasses and quartz slides with distilled water.

      2. Dry the cover glasses and quartz slides by blowing nitrogen gas.

      3. Place the quartz slides on aluminum foil.

      4. Attach double-sided tape to the quartz slides to make channels.

      5. Place a cover glass on each quartz slide.

      6. Block the sides of each channel with epoxy glue.

      7. Insert tubing into the holes and fix them with epoxy glue.



      Figure 3. Flow cell assembly. A. Procedure to prepare the flow cell from a PEGylated slide. B. Flow cell after assembly. Inlet and outlet are connected to the injected solution and syringe pump, respectively.


  3. Single-molecule fluorescence measurement

    1. Preparation

      1. Turn on the devices and set the microscope temperature control system to 37°C.

      2. Drop a small amount of water on the objective lens as water immersion.

      3. Place a fluorescent bead slide on the microscope stage.

      4. Drop a small amount of oil on the bead slide and mount a prism.

      5. Illuminate the bead slide, and focus on it by properly adjusting the distance between the bead slide and the objective lens.

      6. Adjust the illumination area.

      7. Turn on the CCD and adjust the dichroic mirrors to divide the imaging area into three different wavelength ranges.

      8. Measure the signals from the fluorescent beads.

      9. To identify fluorescence signals of different channels from the same molecule, make a reference mapping file with a fluorescent bead image.

        Note: We use three-color alternating-laser excitation to obtain signals from Cy3, Cy5, and Alexa Fluor 488 dye. To do this, we divide the whole CCD image into three equal areas corresponding to different wavelength ranges. Therefore, we need to know the position information of the same molecules in the different detection channels (see Figure 4). This step is crucial in experiments using a labeled antibody, because only signals emitted by co-localization of the antibody with DNA are considered specific binding. The three dyes were selected due to their superior photostabilities and good quantum yields.

      10. Remove the bead slide.



      Figure 4. Fluorescent bead image to obtain the position information of the same molecules. Fluorescent beads are excited by a 473-nm laser, and images are recorded separately according to three different wavelength ranges (Cy3, Cy5, and Alexa Fluor 488 channels). Through Gaussian fitting, the peaks are searched (white circle: co-localized points).


    2. Experiments to observe GQ formation during single-round transcription

      1. Preparation of the stalled elongation complex.

        Note: 11 nucleotides of the non-template strand from the transcription start site consist of only adenine and guanine.

        If T7 RNA polymerase transcribes the DNA without cytosine and thymine, transcription will be stopped with 11-nt-long RNA. This conformation is called the stalled elongation complex. After immobilizing this stalled elongation complex on the surface of the flow cell, we inject rNTP to restart elongation. Using this strategy, we could observe GQ formation under single-round transcription.

        1. Prepare a PCR-size tube with 5 µl distilled water.

        2. Add 2.5 µl 4× elongation buffer, 0.25 µl 1 µM DNA, and 0.25 µl mixture of 2.5 mM GTP and ATP.

        3. Mix by pipetting.

        4. Add 2 µl T7 RNA polymerase.

        5. Mix by pipetting very gently.

        6. Place tube in a thermal cycler and incubate at 37°C.

          Note: The incubation time should not exceed 40 min. Due to the error rate of T7 RNA polymerase in transcription, unexpected RNA with the wrong bases could be created and affect the GQ and R-loop formation efficiency.

      2. Make 600 µl imaging buffer (see Recipes).

      3. Mount the flow cell on the microscope stage.

      4. Connect the outlet of the flow cell to a syringe pump.

      5. Using a syringe pump, wash out the channel with 90 µl T50 buffer more than twice.

      6. Inject 90 µl streptavidin (200 ng/ml) into the channel.

      7. Wash out the unbound streptavidin in the channel with T50 buffer 5 min after injection.

      8. Turn on the CCD and adjust the focus and illumination area.

      9. Add 0.5 µl solution containing the stalled elongation complex to 100 µl imaging buffer and gently mix by pipetting.

      10. Inject 90 µl imaging buffer containing the stalled elongation complex into the channel.

      11. Check whether the number of spots is sufficient at Cy3 excitation.

      12. Wash out the unbound stalled elongation complexes in the channel 3 times with 90 µl imaging buffer.

      13. Add rNTP to the imaging buffer (the final concentration of rNTP should be 2 mM).

      14. Move the microscope stage and search for the proper imaging area in the channel.

      15. Turn on the auto-focusing system.

        Note: The auto-focus in the z-direction uses a real-time optical astigmatism analysis of single-molecule images. The detailed methods can be found in our previous paper (Hwang et al., 2012).

      16. Start the measurement.

      17. Inject the imaging buffer containing rNTP at 25 s after starting measurement.

      18. Measure for 10 min.

      19. Stop the measurement.

    3. Experiments to observe GQ formation during multiple-round transcription

      1. Add 0.25 µl 1 µM DNA to 9.75 µl distilled water and store in a drawer during the experiment.

      2. Make 600 µl imaging buffer (see Recipes).

      3. Mount the flow cell on the microscope stage.

      4. Connect the outlet of a flow cell to a syringe pump.

      5. Using a syringe pump, wash out the channel more than twice with 90 µl T50 buffer.

      6. Inject 90 µl streptavidin (200 ng/ml) into the channel.

      7. Wash out the unbound streptavidin in the channel with T50 buffer 5 min after injection.

      8. Add 0.4 µl 25 nM DNA to 100 µl imaging buffer and gently mix by pipetting.

      9. Inject 90 µl imaging buffer containing DNA into the channel.

      10. Check whether the number of spots is sufficient at Cy3 excitation.

      11. Wash out the unbound DNA in the chamber 3 times with 90 µl imaging buffer.

      12. Add rNTP and T7 RNA polymerase to the imaging buffer (their final concentration should be 2 mM and 8 nM, respectively) and mix by pipetting.

      13. Incubate at 37°C for 5 min.

      14. Move the microscope stage and search for a proper imaging area in the channel.

      15. Turn on the auto-focusing system.

      16. Start the measurement.

      17. Inject the imaging buffer containing rNTP and T7 RNA polymerase at 25 s after starting measurement.

      18. Measure for 30 min. (In the case of a time-lapse experiment, measure for 3 min at each time point. Do not measure the same area to avoid photobleaching.)

      19. Stop the measurement.

    4. Experiments to observe R-loop formation

      1. Add 0.25 µl 1 µM DNA to 9.75 µl distilled water and store in a drawer during the experiment.

      2. Make 600 µl imaging buffer (see Recipes).

      3. Mount the flow cell on the microscope stage.

      4. Connect the outlet of a flow cell to a syringe pump.

      5. Using a syringe pump, wash out the channel with 90 µl T50 buffer more than twice.

      6. Inject 90 µl streptavidin (200 ng/ml) into the channel.

      7. Wash out the unbound streptavidin in the channel with T50 buffer 5 min after injection.

      8. Add 0.4 µl 25 nM DNA to 100 µl imaging buffer and gently mix by pipetting.

      9. Inject 90 µl imaging buffer containing DNA into the channel.

      10. Check whether the number of spots is sufficient at Cy3 excitation.

      11. Wash out the unbound DNA in the chamber 3 times with 90 µl imaging buffer.

      12. Take the S9.6 antibody out the -80°C freezer and store in the -20°C freezer for 10 min.

      13. Thaw the S9.6 antibody in on ice.

      14. Add rNTP and T7 RNA polymerase to the imaging buffer (Their final concentration should be 2 mM and 8 nM, respectively) and mix by pipetting.

      15. Add the S9.6 antibody and labeled secondary antibody to the imaging buffer (Their final concentration should be 33 nM) and mix by pipetting.

      16. Incubate the imaging buffer containing rNTP, T7 RNA polymerase, and labeled antibody at 37°C for 5 min.

      17. Move the microscope stage and search for a proper imaging area in the channel.

      18. Adjust the incident beam intensity of the blue laser.

        Note: It is challenging to know the proper incident beam intensity since there are a few spots (junk) in the Alexa Fluor 488 channel before injecting the labeled antibody into the flow cell. We recommend finding the proper incident beam intensity by injecting antibody into the flow cell where pre-formed DNA-RNA hybrid exists.

      19. Turn on the auto-focusing system.

      20. Start the measurement.

      21. Inject the buffer containing rNTP, T7 RNA polymerase, and labeled antibody at 25 s after starting measurement.

      22. Measure for 30 min (In the case of a time-lapse experiment, measure for 3 min at each time point. Do not measure the same area to avoid photobleaching).

      23. Stop the measurement.

Data analysis

FRET efficiency, E, is defined as the fluorescence intensity of the acceptor (Cy5) relative to the sum of the donor (Cy3) and acceptor (Cy5) intensities at donor excitation. Using the FRET efficiency between Cy3 and Cy5 labeled at the nucleotides flanking the GQ-forming sequence, we could identify DNA conformations formed in the GQ-forming region. Before rNTP and RNAP injection, the low FRET (E = 0.13) indicates that the region remains as a duplex. After the injection of rNTP and RNAP, the FRET efficiency increases to E = 0.82 via an intermediate FRET of E = 0.37 (Figure 5A, top and middle panels). The high FRET state is identified as GQ, but the exact conformation corresponding to the intermediate FRET is not yet known. On the other hand, the R-loop formation is detected by monitoring the binding of the fluorescently labeled S9.6 antibody, which specifically binds to DNA–RNA hybrids (Figure 5A, bottom panel). Note: Non-specific binding of the S9.6 antibody was screened by SNR and binding lifetime criteria).


From a time-lapse experiment, the evolution of the population of each state can be investigated (Figure 5B). Note: Overlapping molecules with a high intensity that deviates from the average intensity of all molecules by 30% are excluded. It is evident that GQ accumulates over time, whereas the intermediate state vanishes. The time delay from GQ formation to the first antibody binding can also be measured. The time delay is mostly negative (Figure 5C), indicating that R-loop formation precedes GQ formation. R-loop formation efficiency can be measured by counting the DNA substrate to which the S9.6 antibody binds. We found that the R-loop formation efficiency is increased up to 5 times when GQ exists on the non-template strand of DNA (Figure 5D), indicating the existence of a positive feedback loop between GQ formation and R-loop formation.



Figure 5. Co-transcriptional formation of GQ coupled with R-loops. A. Representative time traces showing GQ and R-loop formation. GQ: Cy3 (top, green) and Cy5 (top, red) fluorescence intensities at Cy3 excitation, and the corresponding FRET (middle). R-loop: a sudden increase in Alexa Fluor 488 fluorescence intensity at Alexa Fluor 488 excitation (bottom). B. Relative populations of the intermediate state (orange) and GQ state (blue) over time. The population sum of the intermediate state and GQ state (black) is fitted to a single-exponential function with a time constant of 45.3 ± 5.6 min (black lines). C. A histogram of the time difference between GQ formation and the first antibody binding [t1 is defined in the middle panel of A]. D. The portion of dsDNA that is coupled with antibody binding for 20 min after the start of transcription. The R-loop formation efficiency significantly increases when GQ exists on the non-template strand. Figure 5 was adapted from the original paper (Lim and Hohng, 2020).

Recipes

  1. Trolox

    1. Dissolve 50 mg Trolox in 50 ml distilled water

    2. Add 50 µl 3 M NaOH and vortex

    3. Rotate the tube for one day at room temperature (RPM: 30)

    4. Filter the solution with a 0.2-µm membrane filter

    Store at 4°C

  2. PCA

    1. Dissolve 154 mg PCA in 20 ml distilled water

    2. Add 20 µl 3M NaOH and vortex

    3. Filter the solution and store at 4°C

  3. T50 buffer

    Add 500 µl Tris-HCl (pH 8.0) and 500 µl 5 M NaCl to 49 ml distilled water

    Vortex vigorously

  4. Denaturing gel solution

    1. Pour 2.5 ml distilled water into a 15-ml tube containing 4.8 g UREA

    2. Add 0.5 ml 10× TBE buffer

    3. Add 3 ml Acry:Bis=29:1 solution

    4. Vortex vigorously

    5. Warm the solution in a microwave to dissolve the UREA

    6. Cool at 4°C for 15 min

  5. Piranha solution

    1. Pour 90 ml sulfuric acid into a heat-resistant container

    2. Add 30 ml hydrogen peroxide solution (30%) (final ratio of hydrogen peroxide solution to sulfuric acid should be 1:3)

    3. Gently shake

    Note: The solution will boil and be hot after mixing.

  6. Silanization solution

    1 ml aminoprophysilane

    5 ml acetic acid

    100 ml methanol

  7. PEGylation solution

    1. Mix 2 mg biotin-PEG-NHS ester, 80 mg PEG-NHS ester, and 640 µl 100 mM sodium bicarbonate solution

    2. Vortex and centrifuge

    Note: After dissolving, promptly use to avoid degradation.

  8. Imaging buffer

    Mix 65 µl Trolox, 10 µl distilled water, 10 µl PCA, 3 µl PCD, 4 µl 1M Tris-HCl (pH 8.0), 2.5 µl 2 M KCl, 2 µl 1 M MgCl2, and 2 µl 100 mM spermidine

    Note: According to the type of experiment, a portion of distilled water can be replaced with rNTP, RNAP, and antibody.

  9. 4× elongation buffer

    Mix 160 mM Tris-HCl (pH 8.0), 200 mM KCl, 80 mM MgCl2, and 4 mM DTT

    Store at -20°C

Acknowledgments

The National Research Foundation of Korea [NRF-2019R1A2C2005209 to S.H.]. This protocol was derived from the original paper “Single-molecule fluorescence studies on cotranscriptional G-quadruplex formation coupled with R-loop formation,” published in Nucleic Acids Research in 2020.

Competing interests

Nothing to declare.

References

  1. Aguilera, A. and Garcia-Muse, T. (2012). R loops: from transcription byproducts to threats to genome stability. Mol Cell 46(2): 115-124.
  2. Biffi, G., Tannahill, D., McCafferty, J. and Balasubramanian, S. (2013). Quantitative visualization of DNA G-quadruplex structures in human cells. Nat Chem 5(3): 182-186.
  3. Biffi, G., Tannahill, D., Miller, J., Howat, W. J. and Balasubramanian, S. (2014). Elevated levels of G-quadruplex formation in human stomach and liver cancer tissues. PLoS One 9(7): e102711.
  4. Chan, Y. A., Aristizabal, M. J., Lu, P. Y., Luo, Z., Hamza, A., Kobor, M. S., Stirling, P. C. and Hieter, P. (2014). Genome-wide profiling of yeast DNA:RNA hybrid prone sites with DRIP-chip. PLoS Genet 10(4): e1004288.
  5. De Magis, A., Manzo, S. G., Russo, M., Marinello, J., Morigi, R., Sordet, O. and Capranico, G. (2019). DNA damage and genome instability by G-quadruplex ligands are mediated by R loops in human cancer cells. Proc Natl Acad Sci U S A 116(3): 816-825.
  6. Duquette, M. L., Handa, P., Vincent, J. A., Taylor, A. F. and Maizels, N. (2004). Intracellular transcription of G-rich DNAs induces formation of G-loops, novel structures containing G4 DNA. Genes Dev 18(13): 1618-1629.
  7. Falabella, M., Kolesar, J. E., Wallace, C., de Jesus, D., Sun, L., Taguchi, Y. V., Wang, C., Wang, T., Xiang, I. M., Alder, J. K., Maheshan, R., Horne, W., Turek-Herman, J., Pagano, P. J., St Croix, C. M., Sondheimer, N., Yatsunyk, L. A., Johnson, F. B. and Kaufman, B. A. (2019). G-quadruplex dynamics contribute to regulation of mitochondrial gene expression. Sci Rep 9(1): 5605.
  8. Gellert, M., Lipsett, M. N. and Davies, D. R. (1962). Helix formation by guanylic acid. Proc Natl Acad Sci U S A 48: 2013-2018.
  9. Hansel-Hertsch, R., Spiegel, J., Marsico, G., Tannahill, D. and Balasubramanian, S. (2018). Genome-wide mapping of endogenous G-quadruplex DNA structures by chromatin immunoprecipitation and high-throughput sequencing. Nat Protoc 13(3): 551-564.
  10. Hwang, W., Bae, S. and Hohng, S. (2012). Autofocusing system based on optical astigmatism analysis of single-molecule images. Opt Express 20(28): 29353-29360.
  11. Kreig, A., Calvert, J., Sanoica, J., Cullum, E., Tipanna, R. and Myong, S. (2015). G-quadruplex formation in double strand DNA probed by NMM and CV fluorescence. Nucleic Acids Res 43(16): 7961-7970.
  12. Lee, J., Lee, S., Ragunathan, K., Joo, C., Ha, T. and Hohng, S. (2010). Single-molecule four-color FRET. Angew Chem Int Ed Engl 49(51): 9922-9925.
  13. Lim, G. and Hohng, S. (2020). Single-molecule fluorescence studies on cotranscriptional G-quadruplex formation coupled with R-loop formation. Nucleic Acids Res 48(16): 9195-9203.
  14. Marsico, G., Chambers, V. S., Sahakyan, A. B., McCauley, P., Boutell, J. M., Antonio, M. D. and Balasubramanian, S. (2019). Whole genome experimental maps of DNA G-quadruplexes in multiple species. Nucleic Acids Res 47(8): 3862-3874.
  15. Masse, E. and Drolet, M. (1999). Escherichia coli DNA topoisomerase I inhibits R-loop formation by relaxing transcription-induced negative supercoiling. J Biol Chem 274(23): 16659-16664.
  16. Roy, D. and Lieber, M. R. (2009). G clustering is important for the initiation of transcription-induced R-loops in vitro, whereas high G density without clustering is sufficient thereafter. Mol Cell Biol 29(11): 3124-3133.
  17. Roy, R., Hohng, S. and Ha, T. (2008). A practical guide to single-molecule FRET. Nat Methods 5(6): 507-516.
  18. Santos-Pereira, J. M. and Aguilera, A. (2015). R loops: new modulators of genome dynamics and function. Nat Rev Genet 16(10): 583-597.
  19. Sanz, L. A., Hartono, S. R., Lim, Y. W., Steyaert, S., Rajpurkar, A., Ginno, P. A., Xu, X. and Chedin, F. (2016). Prevalent, Dynamic, and Conserved R-Loop Structures Associate with Specific Epigenomic Signatures in Mammals. Mol Cell 63(1): 167-178.
  20. Siddiqui-Jain, A., Grand, C. L., Bearss, D. J. and Hurley, L. H. (2002). Direct evidence for a G-quadruplex in a promoter region and its targeting with a small molecule to repress c-MYC transcription. Proc Natl Acad Sci U S A 99(18): 11593-11598.

简介

[摘要] G-四ES (GQ)和R-环小号是与基因的非规范核酸结构REG UL通货膨胀和基因组不稳定性的是可转录过程中形成; 然而,它们的形成机制仍然难以捉摸。到地址日是问题,我们开发了一个单molec UL ë荧光技术来监测G-四链体和R-环的形成结构转录过程。使用这种技术,我们发现 R 环的形成先于 GQ 的形成,并且 G-四链体和 R 环的形成之间存在正反馈环。

[背景] G-四ES (GQ)由堆叠的G-四联体的小号被形成从4的Hoogsteen碱基配对的鸟嘌呤(盖勒特等人,1962) 。GQ形成的某些热点的基因组(BIFFI等人,2013;的Marsico等,2019)发挥REG UL atory角色(西迪基,耆那教等,2002;法拉贝拉等人,2019)和被相关,以一定的疾病(Biffi等人,2014 年;De Magis等人,2019 年)。R-环s的三链核酸结构小号双链DNA-RNA杂交和单链DNA组成。R-环小号都还涉及到基因REG UL通货膨胀和基因组不稳定性(斯蒂娜和加西亚-缪2012;桑托斯-Pereira的和Aguilera的2015) 。有趣的是,GQ和R-环小号的转录过程中形成的,而且往往共存(杜奎特等人,2004) 。

在B GQ和R-环iological角色小号及其形成机制已经在过去的几十年中得到了广泛的研究。GQ和R-环形成已先前被使用双检测UL FITE测序(Roy和利伯2009)或immunoprecipitatio后测序Ñ (陈等人,2014;汉塞尔-Hertsch等人,2018) 。与GQ 特异性结合的荧光配体也已用于观察 GQ 的形成(Kreig等,2015)。这些技术提供了其中GQ和R-环的热点理解小号通常形成(桑斯等人,2016) ,除了酶REG UL吃GQ的形成和R-环小号(马塞和Drolet 1999 ) ; 然而,该IR确切形成机制仍有待阐明。

这里,我们报告单molec UL Ë荧光监测GQ和R-环路的cotranscriptional形成技术小号。为了检测 GQ 的形成,我们使用了 FRET(荧光共振能量转移),为了检测R 环的形成,我们使用了荧光标记的 S9.6抗体(图 1)。我们在转录过程中监测了 GQ 和 R-loop 的形成,发现 R-loop 的形成先于 GQ 的形成,并且 GQ 和 R-loop 的形成之间存在正反馈环。


图 1. 单分子荧光技术监测GQ 与 R-loop s的共转录形成。GQ 和 R 环的形成分别由高 FRET 效率和标记的 S9.6抗体监测。

关键字:单分子荧光, G-四链体, R环, FRET, 转录

材料和试剂

1.双-双面胶带(3M公司,目录号:137-ROK)     

2.聚乙烯管(BD Intramedic ,Fisher Scientific公司,目录号:427411)     

3.石英显微镜载玻片(H. FINKENBEINER Inc.,美国,目录号:定制订单,尺寸:1'' × 3'' × 1mm)     

4.微盖玻璃(VWR,USA,目录号:48393230)     

5. DNA 寡核苷酸(图 2)     

非模板链(1/2;购自 IDT):
ATCAGGTCTAATACGACTCACTATAGGAAGAGAAAGT/iCy5/TTCTGGGAGGG


非模板链(2/2;购自 IDT):
/ 5 PHOS / AGGGAGGGTGTA / iCy3 / CTGATGCGTTCCACTCGC


模板链(1/1;购自 IDT):
GCGAGTGGAACGCATCAGTACACCCTCCCTCCCTCCCAGAAACTTTCTCTTCCTATAGTGAGTCGTATTAGACCTGAT/生物素/




图 2. 示例设计。DNA 底物是通过退火和连接(顶部)制成的。DNA 底物包含一个T7 启动子和一个GQ 形成序列。生物素(粉红色)标记在上游端用于表面固定,Cy3(绿色)和 Cy5(红色)标记为 FRET 对以检测 GQ 形成。


6.蒸馏水     

7.液氮     

8.丙烯酰胺:bis =29:1(30%)(Biosesang ,目录号:A2003-1)     

9. 10 × TBE(Biosesang ,目录号:TR2004-100-00)     

10. N,N,N”,N'-吨etra-甲基乙二胺(TEMED)(B IO -R广告,目录号:161-0801) 

11.铵个人UL命运(APS)(Sigma-Aldrich公司,目录号:1001799334) 

12.甲酰胺(Sigma-Aldrich,目录号:01246) 

13.尿素(B io -R ad ,目录号:161-0731) 

14.乙醇(C阿洛Ë胸罩ř eagents ,目录号:528167) 

15. Alexa Fluor 488 C5-马来酰亚胺(Thermo Fisher,Invitrogen,目录号:A10254) 

16. NAP-5-柱(GE Healthcare,目录号:17-0853-02) 

17.第一抗体:抗DNA - RNA ħ YBRID(S9.6)一个ntibody(Kerafast ,目录号:ENH001) 

注意:我们把S9.6的原液抗体成等分试样,冷冻各管在液氮,一个他们第二储存在-20 ℃(短期)或-80℃(长期)。


18.二抗:抗小鼠IgG(H+L)(Jackson ImmunoResearch ,目录号:715-005-151) 

19.小号UL furic一个CID(Daejung ,目录号:7664-93-9) 

20.氢p eroxide溶液(30%)(DaeJung ,目录号:7722-84-1) 

21.生物素-PEG-SC(Laysan Bio Inc.,目录号:141-63) 

22. mPEG -SVA(Laysan Bio Inc.,目录号:149-75) 

23.环氧胶(LOCTITE,目录号:326795) 

24. T4 DNA升igase(新英格兰生物实验室,目录号:M0202M) 

25. 10 × T4 DNA 连接酶缓冲液(New England Bio Labs,目录号:B0202S) 

26. T7 RNA聚合酶(New England Bio Labs,目录号:M0251S) 

27. Trolox(Sigma-Aldrich,目录号:238813) 

28. PCD(东方酵母有限公司,目录号:46852004) 

29. PCA(Sigma-Aldrich,目录号:37580) 

30.亚精胺(Sgima- Aldrich,目录号:85558) 

注意:只有一个小用量与在一周稀释,并存储在一个抽屉从该光被完全阻挡。使用原液后,应将容器内的空气用氮气置换,并在室温下保存。


31. rNTP的组(GE ħ ealthcare,目录号:27202501 ) 

32. 1,4- d硫苏糖醇(默克,目录号:222-468-7) 

注意:众所周知,DTT 在水中很容易随着时间的推移而被氧化。为防止降解,请将 1 M DTT 分成等分试样,并在溶解后将它们储存在 -20 °C 。此外,即使 DTT 溶液已在-20 °C 下储存,也建议在制作后几个月不要使用 DTT 溶液。

33.乙酸一个CID(JTBaker ,费舍尔科学,目录号:14-650-388) 

34.钠b icarbonate(Fisher Scientific公司,目录号:S233-500) 

35.钠b硼酸盐(Fisher Scientific公司,目录号:S248-500) 

36.氨基丙基硅烷(UCT SPERCIALTIES,LLC,目录号:A0700) 

37.荧光珠小号(FluoSpheres ,Invitrogen,目录号:1890851) 

38. 10 × TBE 缓冲液(Biosesang ,目录号:TR2004-100-00) 

39. PCR纯化试剂盒(QIAGEN,目录号:28106) 

40. Trolox溶液(见配方) 

41. PCA解决方案(见食谱) 

42. T50 缓冲液(见配方) 

43.变性凝胶溶液(见配方) 

44.食人鱼解决方案(见食谱) 

45.硅烷化溶液(见配方) 

46.聚乙二醇化溶液(见配方) 

47.成像缓冲液(见食谱) 

48. 4    ×延伸缓冲液(见配方)


设备


自制 TIRF 显微镜设置
注意:在使用的组分的自制全内反射荧光(TIRF)显微镜系统如下:EMCCD,显微镜,激光器(473纳米,532纳米,633纳米),快门控制器,棱镜,分色镜,压电载物台和显微镜温度控制系统。详细的实验设置可以在以前发表的论文中找到(Roy 等人,2008 年;Lee 等人,2010 年)。


显微镜温度控制系统(现场ç ELL我nstrument,米Odel等:CU-109)
培养箱(FINE PCR, 米Odel等:ALB 6400)
热循环仪(B IO -R广告,米Odel等:C1000)
垂直电泳细胞(迷你PROTEAN)(B IO -R广告,米Odel等:1658000 FC)
凝胶文档系统(SYNGENE,米Odel等:G:框CHEMI XT4系统)
UL trasonic清洁器(用作水浴)(布兰森,米Odel等:3510E-DTH)
注意:超声波清洗机在加热模式下可用作水浴。我们将温度设置为 69 °C,但运行变性凝胶时的实际温度约为 55°C。


剃须刀(韩国Dorco )
金刚石实体薄钻(OD:0.75 mm)(UKAM Industrial Superhard Tools,目录号:2040075)
玻璃罐(食人鱼用)
水净化系统(Millipore,米Odel等:ZRXQ-003-EU)
注射泵(HARVARD APPARATUS,米Odel等:PHD2000)
分光光度计(Thermo Scientific的,米奥德尔:NANODROP 2000)


软件


Labview(版本:2015)(National Instruments,美国,https://www.ni.com/ )
IDL(版本:7.0)(David Stern 和 ITT Visual Information Solutions,美国,https://www.l3harrisgeospatial.com/Software-Technology/IDL)
Matlab(版本:R2015a)(MathWorks,美国,https ://www.mathworks.com )
Origin(版本:8.5)(Electronic Arts,美国,https://www.origin.com)


程序


样品制备
DNA连接
退火
制备PCR-大小管是兼容的使用的一个热循环- [R 。
通过移液将 4 µl每个100 µM DNA 寡核苷酸混合在 T50 缓冲液中(最终浓度:5 µM,体积:80 µl)。
将包含所述管的在热循环仪的寡核苷酸。
在 95 °C 下将溶液孵育 3 分钟。
将溶液从 80 °C缓慢冷却至 25 °C 。甲-1冷却速度℃下每分钟SH乌尔德是足够的。
退火后将热循环仪的温度保持在 4°C。
结扎
转移退火的DNA至1.7 -毫升管。
加入 60 µl 蒸馏水。
添加 16 µl 10 × T4 连接酶缓冲液。
通过移液混合溶液。
加入 4 µl 5 × T4 DNA 连接酶(溶液总体积:160 µl)。
通过移液混合溶液并储存在保持在 16 °C的孵化器中 16 小时。
微升的5M NaCl和560微升乙醇添加80到所述160微升溶液(结扎后)和通过移液混合。
在-20 °C 下孵育溶液2 小时以上。
离心该溶液在4 ℃下(RCF :16 ,100 ×克,和使用平衡器)。
移除缓冲区。一定不要吸了沉淀。
通过在室温下将盖子打开在抽屉内30 分钟来干燥颗粒,以消除任何剩余的 EtOH。
将沉淀溶解在 4 µl 蒸馏水中并加入 4 µl 99% 甲酰胺。
纯化
制备的凝胶浇铸室(1 -毫米厚)和洗涤用蒸馏水。
制作变性凝胶(见食谱)。
加入 7 µl TEMED 和 60 µl 20% APS 并充分混合。
注意:当添加荷兰国际集团TEMED和APS,所述变性凝胶溶液应该冷静足以防止急剧凝固倒入之前的铸造腔室中。


将溶液倒入凝胶板组的空隙中并插入梳子。
等到凝胶已经完全solidif灭蝇灯。
组装内腔并安装在外腔中。
制作 0.5 × TBE 缓冲液并倒入内室和外室s 。
P花边外室在一保持在55热浴℃。
在预运行之前,通过移液清洗孔。
运行在200伏的凝胶而没有任何样品负荷的ED超过30分钟。
停止预运行并通过移液再次清洗孔。
在每个孔中加载 8 µl 样品。
在 200 V 下再次运行凝胶 40 分钟。
停止运行凝胶并采取凝胶的图片的红色下-色版灯。
使用剃须刀仅提取顶部的第一条带。
注:其他频带中的第一频带对应于下面的DNA片段的是不正确连接。


制备的凝胶纯化试剂盒和对花边一列中的凝胶切片。
将 200 µl 蒸馏水倒入柱中,室温孵育 16 小时。
恢复和再退火
将纯化试剂盒以 16 , 100 × g离心1 分钟。
收集 180 µl 纯化的 DNA,并与 90 µl 5 M NaCl 和 630 µl EtOH 混合。
在-20 °C 下储存2 小时。
在 4 °C 下离心40 分钟。
去除溶液。
将打开的管在抽屉中干燥30 分钟,以消除任何剩余的乙醇。
将沉淀溶解在10 µl 蒸馏水中,并与 0.2 µl 1 M LiCl 和 0.4 µl 1 M Tris-HCl (pH 8.0) 混合。
注意:w ^ ë期间使用的LiCl代替氯化钠以防止G-四链形成的退火工艺。


转移到PCR -大小管。
将管置于热循环仪中。
在 95 °C 下孵育 3 分钟
将样品从 80 °C缓慢冷却至 25 °C 。甲-1冷却速度℃下每分钟是足够的。
储存在-20°C。
抗体标记
将 1 mg 马来酰亚胺A lexa Fluor 488 染料溶解在 55 µl DMSO 中。
在 100 mM 碳酸氢钠溶液中混合 50 µl 抗体和 2 µl 染料。
在室温下将混合物孵育 2 小时。
消除未反应的染料通过纯化使用的NAP5柱。
使用分光光度计检查标记效率。
将标记的抗体储存在 4 °C。


流动池制备
打孔
使用金刚石钻头在石英滑块上打孔(图 3)。


注意:如果大小的孔过大,环氧树脂胶可以泄漏到流动单元的内部空间中。为了防止这种情况,要的尺寸比较是很重要的孔与直径的的钻探过程中PE管材。


PEG涂层
打扫
P花边盖玻璃和石英载玻片装入玻璃罐。
用蒸馏水冲洗盖玻片和石英载玻片数次。
倾Piranha溶液(见配方)插入所述罐和孵育为20 - 30分钟,以清洗所述盖玻璃和石英载玻片。
重复步骤 iii 3 次。
冲洗的盖玻璃和石英片用蒸馏水多次。
硅烷化
用甲醇冲洗干净的盖玻片和石英载玻片两次。
倒入硅烷化解决方案(见食谱)进入该罐。
除去后的溶液一个30 -分钟反应。
冲洗的盖玻片并用甲醇石英载玻片,随后用蒸馏水。
取下的盖玻璃和石英载玻片从所述通过吹入氮气罐和干燥。
聚乙二醇化
制作聚乙二醇化溶液(参见配方)。
将 70 µl PEGylation 溶液滴在石英载玻片上。
P花边玻璃盖在上面。
在抽屉中孵育 4 小时。 
孵育后储存在-20 °C。
流通池组件(图 3)
用蒸馏水解冻和冲洗聚乙二醇化盖玻片和石英载玻片。
吹氮气吹干盖玻片和石英载玻片。
将石英幻灯片s放在铝箔上。
连接双-双面胶带,以将石英滑动š使信道。
在每个石英载玻片上放置一个盖玻片。
用环氧树脂胶堵住每个通道的侧面。
将管子插入孔中并用环氧树脂胶固定它们。




图 3. 流通池组件。A. 从聚乙二醇化载玻片制备流动槽的程序。B. 组装后的流通池。入口和出口分别连接到注射溶液和注射泵。


单molec UL É荧光测量
准备
打开设备并将显微镜温度控制系统设置为37 °C 。
在物镜上滴少量水作为浸水。
将荧光珠载玻片放在显微镜载物台上。
在珠滑梯上滴少量油并安装棱镜。
照亮珠滑,并通过适当调整珠滑与物镜之间的距离来聚焦。
调整所述照明区域。
打开的CCD和调整所述分色镜至所述划分成像区域成三个不同的波长范围。
测量从信号的荧光珠小号。
为了识别来自同一molec不同信道的荧光信号UL E,使用荧光珠图像的参考映射文件。
注意:我们使用三色交替激光激发从 Cy3、Cy5 和 Alexa Fluor 488 染料中获取信号。要做到这一点,我们把在整个CCD图像分成三个相等的区域对应荷兰国际集团到不同的波长范围。因此,我们需要知道相同分子在不同检测通道中的位置信息(见图4)。这一步是在实验关键小号使用标记的抗体,因为只有通过的共定位发射的信号的与DNA抗体被认为是特异性结合。选择这三种染料是因为它们具有优异的光稳定性和良好的量子产率。


取下珠滑梯。




图4.荧光珠图像获得相同molec的位置的信息的UL ES。荧光珠通过激发一个473-nm激光和图像根据三个不同的波长范围内分开记录小号(CY3,CY5,和Alexa的氟488个信道)。通过高斯拟合,搜索峰值(白色圆圈:共定位点)。


在单轮转录过程中观察 GQ 形成的实验
的制备的停滞延伸复合物。
注:11个核苷酸的非模板链的转录起始位点仅由腺嘌呤和鸟嘌呤。


如果T7 RNA聚合酶录制Š的DNA,而不胞嘧啶和胸腺嘧啶,转录将与11-nt的长度的RNA被停止。这种结构被称为在停滞延伸复合物。将这种停滞的伸长复合物固定在流动池表面后,我们注入rNTP以重新开始伸长。使用这种策略,我们可以在单轮转录下观察 GQ 的形成。


用 5 µl 蒸馏水准备一个PCR 大小的管。
加入 2.5 µl 4 ×延伸缓冲液、0.25 µl 1 µM DNA 和 0.25 µl 2.5 mM GTP 和 ATP 的混合物。
通过移液混合。
添加 2 µl T7 RNA 聚合酶。
非常轻柔地移液混合。
将管置于热循环仪中并在 37°C 下孵育。
注:在我ncubation时间不应超过40分钟。由于T7 RNA的误差率在转录聚合酶,意想不到的RNA中可以创建错误的基础和影响GQ和R-循环形成效率。


制作 600 µl 成像缓冲液(参见食谱)。
将流动槽安装在显微镜载物台上。
将流通池的出口连接到注射泵。
使用注射泵,用 90 µl T50 缓冲液冲洗通道两次以上。
将 90 µl 链霉亲和素 (200 ng/ ml ) 注入通道。
注射后 5 分钟,用 T50 缓冲液清洗通道中未结合的链霉亲和素。
打开的CCD和调整焦距和照明区域。
添加0.5μl的溶液含有的停滞延伸复合物至100μl成像缓冲器和绅士LY通过移液混合。
注射90微升成像缓冲器包含的停滞延伸复合物到通道中。
检查Cy3 激发时点的数量是否足够。 
用 90 µl 成像缓冲液清洗通道中未结合的停滞延伸复合物3 次。
将 rNTP 添加到成像缓冲液中(rNTP 的最终浓度应为 2 mM)。
移动显微镜载物台,并搜索用于在信道适当成像区。
打开自动对焦系统。
注意:所述的自动聚焦在所述z方向上使用单分子图像的实时光学散光分析。在详述的方法Ç一个在我们以前的发现纸(Hwang等人,2012) 。


开始的测量。
在开始测量后 25 秒注入含有 rNTP 的成像缓冲液。
中号easure 10分钟。
停止的测量。
在多轮转录过程中观察 GQ 形成的实验
在实验过程中,将 0.25 µl 1 µM DNA 添加到 9.75 µl 蒸馏水中并储存在抽屉中。
制作 600 µl 成像缓冲液(参见食谱)。
将流动槽安装在显微镜载物台上。
将流动池的出口连接到注射泵。
使用注射泵,用 90 µl T50 缓冲液冲洗通道两次以上。
将 90 µl 链霉亲和素 (200 ng/ ml ) 注入通道。
注射后 5 分钟,用 T50 缓冲液清洗通道中未结合的链霉亲和素。
添加0.4微升25 nM的DNA到100μl成像缓冲器和绅士LY通过移液混合。
将含有 DNA 的 90 µl 成像缓冲液注入通道。
检查Cy3 激发时点的数量是否足够。 
用 90 µl 成像缓冲液清洗腔室中未结合的 DNA 3 次。
添加的rNTP和T7 RNA聚合酶到成像缓冲液(其最终浓度昭UL d为2毫米和8 nM的,分别)和通过移液混合。
在 37 °C 下孵育5 分钟。
移动显微镜载物台,并搜索用于在所述通道中的适当的成像区。
打开自动对焦系统。
开始的测量。
在开始测量后 25 秒注入含有rNTP 和 T7 RNA 聚合酶的成像缓冲液。
中号easure 30分钟(在该时间推移实验的情况下,测量在每个3分钟时间点,不可测量相同的区域,以避免光漂白)。
停止的测量。
观察 R 环形成的实验
在实验过程中,将 0.25 µl 1 µM DNA 添加到 9.75 µl 蒸馏水中并储存在抽屉中。
制作 600 µl 成像缓冲液(参见食谱)。
将流动槽安装在显微镜载物台上。
将流动池的出口连接到注射泵。
使用注射泵,用 90 µl T50 缓冲液冲洗通道两次以上。
将 90 µl 链霉亲和素 (200 ng/ ml ) 注入通道。
注射后 5 分钟,用 T50 缓冲液冲洗通道中未结合的链霉亲和素。
添加0.4微升25 nM的DNA到100μl成像缓冲器和绅士LY通过移液混合。
将含有 DNA 的 90 µl 成像缓冲液注入通道。
检查Cy3 激发时点的数量是否足够。 
用 90 µl 成像缓冲液清洗腔室中未结合的 DNA 3 次。
取的S9.6抗体出的-80 ℃的冰箱中,并存储在所述-20℃冷冻机中10分钟。
解冻的S9.6抗体在上冰。
添加的rNTP和T7 RNA聚合酶到成像缓冲液(其最终浓度昭UL d为2毫米和8 nM的,分别)和通过移液混合。
添加的S9.6抗体和标记的第二抗体于成像缓冲液(其终浓度昭UL d是33纳米),并通过移液混合。
孵育成像缓冲器包含的rNTP ,T7 RNA聚合酶,和标记的抗体,在37 ℃下5分钟。
移动显微镜载物台,并搜索用于在所述通道中的适当的成像区。
调整入射光束强度的蓝色激光。
注意:这是具有挑战性知道的适当的入射光束强度,因为有一个几个景点(垃圾)中所述的Alexa氟488信道注入所述标记的抗体到流动池之前。我们建议通过将抗体注入存在预先形成的 DNA - RNA 杂交体的流动池中来找到合适的入射光束强度。


打开自动对焦系统。
开始的测量。
在开始测量后 25 秒注入含有rNTP、T7 RNA 聚合酶和标记抗体的缓冲液。
中号easure 30分钟(在该时间推移实验的情况下,测量在每个3分钟时间点,不可测量相同的区域,以避免光漂白)。
停止的测量。


数据分析


FRET 效率E定义为受体 (Cy5) 的荧光强度相对于供体激发时的供体 (Cy3) 和受体 (Cy5) 强度之和。使用标记的核苷酸侧翼的GQ Cy3和Cy5之间的FRET效率-形成序列,我们共同UL ð识别形成于所述GQ DNA构象-形成区域。在 rNTP 和 RNAP 注射之前,低 FRET ( E = 0.13) 表明该区域仍为双链体。注射rNTP和 RNAP 后,FRET 效率通过E = 0.37的中间 FRET增加到E = 0.82 (图 5A,顶部和中间面板)。高 FRET 状态被确定为 GQ,但与中间 FRET 对应的确切构象尚不清楚。在另一方面中,R-环形成是通过监测荧光的结合来检测标记的S9.6抗体,其特异性结合到DNA - RNA杂交小号(图5A,下图)。注意:非特异性结合的S9.6抗体瓦特如由SNR和结合寿命条件)筛选。


  从时间推移的实验中,在弹出的演变UL每个状态的通货膨胀可以调查(图5B) 。注意:Overlapp荷兰国际集团的分子具有高强度的那个偏离小号从30%的所有分子的平均强度被排除在外。显而易见的是,GQ ACCUM UL茨随着时间的推移,而中间状态消失。也可以测量从 GQ 形成到第一抗体结合的时间延迟。时间延迟大多为负(图 5C),表明 R 环形成先于 GQ 形成。R 环形成效率可以通过对S9.6抗体结合的 DNA 底物进行计数来测量。我们发现,当 GQ 存在于DNA的非模板链上时,R 环形成效率提高了 5 倍(图 5D),表明GQ形成和 R 环形成之间存在正反馈环。




图 5. GQ 与 R-loop s 的共转录形成。A. 显示 GQ 和 R 环形成的代表性时间轨迹。GQ:Cy3 激发下的 Cy3(顶部,绿色)和 Cy5(顶部,红色)荧光强度,以及相应的 FRET(中间)。R-循环:突然增加在Alexa的氟在Alexa488的荧光强度氟488激发(底部)。B.相对弹出UL随时间的中间状态(橙色)和GQ状态(蓝色)的ations。弹出UL中间状态和GQ状态(黑色)的通货膨胀总和被装配到与45.3±5.6分钟(黑线)的时间常数的单指数函数。GQ形成和之间的时间差的C.直方图的第一抗体的结合[T1处于A的中间面板中定义]。D.转录开始后与抗体结合 20 分钟的 dsDNA 部分。当 GQ 存在于非模板链上时,R-环形成效率显着增加。图5是适于FR ö米原纸(Lim和Hohng ,2020年)。


食谱


特洛克斯
溶解50毫克Ť rolox在50ml蒸馏水
加入 50 µl 3 M NaOH 并涡旋
在室温下旋转试管一天 (RPM: 30)
过滤与所述溶液一个0.2μm的膜过滤器
S储存在 4 °C


主成分分析
将 154 毫克 PCA 溶解在 20 毫升蒸馏水中
加入 20 µl 3M NaOH 并涡旋
过滤溶液并储存在 4 °C
T50缓冲液
将 500 µl Tris-HCl (pH 8.0) 和 500 µl 5 M NaCl 添加到 49 ml 蒸馏水中


涡v igorously


变性凝胶溶液
倾2.5毫升蒸馏水成一个15 -含有4.8克UREA毫升管
添加 0.5 ml 10 × TBE 缓冲液
添加 3 ml Acry:Bis =29:1 溶液
涡v igorously
温热该溶液在一个微波炉,以溶解所述UREA
冷却在4℃下15分钟
食人鱼解决方案
将 90 毫升硫酸倒入耐热容器中
加入30毫升双氧水(30%)(双氧水与硫酸的最终比例应为1:3)
根特LY摇
注意:混合后溶液会沸腾并变热。


硅烷化溶液
1毫升氨基丙硅烷


5毫升醋酸


100 毫升甲醇


聚乙二醇化溶液
混合 2 mg 生物素-PEG-NHS 酯、80 mg PEG-NHS 酯和640 µl 100 mM 碳酸氢钠溶液
涡旋和离心机
注意事项:溶解后应及时使用,以免降解。


成像缓冲器
混合65微升Ť rolox,10微升蒸馏水10微升PCA,3μl的PCD,4微升1M的Tris-HCl(pH为8.0),2.5微升的2M的KCl ,2微升的1M的MgCl 2 ,和2微升100mM亚精胺


注:根据实验类型,部分蒸馏水可用rNTP 、RNAP 、抗体代替。


4 ×延伸缓冲液
混合 160 mM Tris-HCl (pH 8.0)、200 mM KCl 、80 mM MgCl 2和 4 mM DTT


储存在-20 °C


致谢


在韩国国家研究基金会[NRF-2019R1A2C2005209到SH] 。该协议从原始纸张“单molec衍生UL Ë荧光上加上R-环形成cotranscriptional G-四链体形成的研究,”发布编在核酸研究,2020年。


利益争夺


无须报关。


参考


Aguilera, A. 和 Garcia-Muse, T. (2012)。R 环:从转录副产品到对基因组稳定性的威胁。摩尔细胞46(2):115-124。
Biffi, G.、Tannahill, D.、McCafferty, J. 和 Balasubramanian, S.(2013 年)。人类细胞中 DNA G-四链体结构的定量可视化。国家化学5(3):182-186。              
Biffi, G.、Tannahill, D.、Miller, J.、Howat, WJ 和 Balasubramanian, S.(2014 年)。人胃癌和肝癌组织中 G-四链体形成水平升高。PLoS 一号9(7):e102711。
Chan, YA, Aristizabal, MJ, Lu, PY, Luo, Z., Hamza, A., Kobor, MS, Stirling, PC 和 Hieter, P. (2014)。使用 DRIP 芯片对酵母 DNA:RNA 杂合位点进行全基因组分析。PLoS 基因10(4):e1004288。              
De Magis, A., Manzo, SG, Russo, M., Marinello, J., Morigi, R., Sordet, O. 和 Capranico, G. (2019)。G-四链体配体引起的 DNA 损伤和基因组不稳定性由人类癌细胞中的 R 环介导。Proc Natl Acad Sci USA 116(3): 816-825。
Duquette, ML, Handa, P., Vincent, JA, Taylor, AF 和 Maizels, N. (2004)。富含 G 的 DNA 的细胞内转录诱导 G 环的形成,即含有 G4 DNA 的新结构。基因开发18(13):1618-1629。              
Falabella, M., Kolesar, JE, Wallace, C., de Jesus, D., Sun, L., Taguchi, YV, Wang, C., Wang, T., Jiang, IM, Alder, JK, Maheshan, R ., Horne, W., Turek-Herman, J., Pagano, PJ, St Croix, CM, Sondheimer, N., Yatsunyk, LA, Johnson, FB 和 Kaufman, BA (2019)。G-四链体动力学有助于调节线粒体基因表达。科学报告9(1): 5605。              
Gellert, M.、Lipsett, MN 和 Davies, DR (1962)。鸟苷酸形成螺旋。Proc Natl Acad Sci USA 48:2013-2018。
Hansel-Hertsch, R.、Spiegel, J.、Marsico, G.、Tannahill, D. 和 Balasubramanian, S.(2018 年)。通过染色质免疫沉淀和高通量测序对内源性 G-四链体 DNA 结构进行全基因组定位。国家议定书 13(3): 551-564。              
Hwang, W.、Bae, S. 和 Hohng, S. (2012)。基于单分子图像光学散光分析的自动对焦系统。选择 Express 20(28):29353-29360。              
Kreig, A.、Calvert, J.、Sanoica, J.、Cullum, E.、Tipanna, R. 和 Myong, S.(2015 年)。通过 NMM 和 CV 荧光探测双链 DNA 中 G-四链体的形成。核酸研究43(16):7961-7970。              
Lee, J.、Lee, S.、Ragunathan, K.、Joo, C.、Ha, T. 和 Hohng, S. (2010)。单分子四色 FRET。Angew Chem Int Ed Engl 49(51): 9922-9925。
Lim, G. 和 Hohng, S.(2020 年)。对共转录 G-四链体形成与 R-环形成相结合的单分子荧光研究。核酸研究48(16):9195-9203。
Marsico, G., Chambers, VS, Sahakyan, AB, McCauley, P., Boutell, JM, Antonio, MD 和 Balasubramanian, S. (2019)。多个物种中 DNA G-四链体的全基因组实验图谱。核酸研究47(8): 3862-3874。              
Masse, E. 和 Drolet, M. (1999)。大肠杆菌DNA 拓扑异构酶 I 通过放松转录诱导的负超螺旋来抑制 R 环的形成。J Biol Chem 274(23):16659-16664。
Roy, D. 和 Lieber, MR (2009)。G 聚类对于体外转录诱导的 R 环的启动很重要,而此后没有聚类的高 G 密度就足够了。Mol Cell Biol 29(11): 3124-3133。              
Roy, R.、Hohng, S. 和 Ha, T. (2008)。单分子 FRET 实用指南。Nat 方法5(6):507-516。
Santos-Pereira, JM 和 Aguilera, A. (2015)。R 环:基因组动力学和功能的新调节剂。Nat Rev Genet 16(10): 583-597。              
Sanz, LA, Hartono, SR, Lim, YW, Steyaert, S., Rajpurkar, A., Ginno, PA, Xu, X. 和 Chedin, F. (2016)。普遍的、动态的和保守的 R 环结构与哺乳动物的特定表观基因组特征相关。摩尔细胞63(1):167-178。
Siddiqui-Jain, A., Grand, CL, Bearss, DJ 和 Hurley, LH (2002)。启动子区域中 G-四链体及其靶向小分子以抑制 c-MYC 转录的直接证据。Proc Natl Acad Sci USA 99(18): 11593-11598。
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引用:Lim, G. and Hohng, S. (2021). Single-molecule Fluorescence Technique to Monitor the Co-transcriptional Formation of G-quadruplex and R-loop Structures. Bio-protocol 11(13): e4069. DOI: 10.21769/BioProtoc.4069.
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