Probing The Structure And Dynamics Of Nucleosomes Using Atomic Force Microscopy Imaging

1. Continuous Dilution Assembly of Mono-nucleosomes

1. Generate and purify an approximately 400 bp DNA substrate that contains an off-centered Widom 601 nucleosome positioning sequence.⁵⁵
   NOTE: To limit the unwanted formation of di-nucleosomes, each ‘arm’ flanking the positioning sequence should not exceed ~150 bp.
   1. Use plasmid pGEM3Z-601 along with the designed primers and amplify the substrate DNA using PCR. For the 423 bp substrate with 122 and 154 bp arm lengths used here, use forward (5’-CAGTGAATTGTAATACGACTC-3’) and reverse (5’-ACAGCTATGACCATGATTAC-3’) primers.
   2. Add tubes containing the reaction mixture to a thermal cycler preheated to 95 °C. Run the following program for 33 cycles after an initial denaturation for 5 min at 95 °C: 30 s denaturation at 95 °C, 30 s annealing at 49 °C, 35 s extension at 72 °C. Set a final extension at 72 °C for 10 min following the 33 cycles.
   3. Purify the DNA from the PCR mixture using a commercially available PCR Purification Kit. When eluting the DNA from the PCR cleanup column, use 10 mM Tris buffer (pH 7.5) in place of the kit provided elution buffer.
      NOTE: Take extra care not to transfer buffers between the purification steps. A contaminated eluent can cause issues downstream when measuring DNA concentration and/or can alter the starting salt concentration of the nucleosome assembly mixture.
2. Determine the DNA concentration by measuring the absorbance of purified DNA at 260 nm.
   1. Collect a blank on the UV VIS Spectrophotometer using only the 10 mM Tris pH 7.5 elution buffer. Collect a measurement of the purified DNA.
3. With the concentration determined, aliquot 25 pmol of the purified DNA into a 0.6 mL microfuge tube and place it in a vacuum centrifuge until the solution is barely visible; this is typically 30 min to 1 h.
   NOTE: The DNA substrate is now ready for nucleosome assembly. Otherwise, the protocol can be paused here and the DNA stored at -20 °C until use.
4. Place the microfuge tube containing the 25 pmol DNA on the ice and add the nucleosome assembly components in Table 1, in the order listed. When all components have been added, remove the mixture from the ice and incubate at room temperature (RT) for 30 min.
   NOTE: It is critical that the salt content of the stock histone buffer is considered when calculating the NaCl needed to achieve the 2 M final concentration.^15,56
5. Assemble the nucleosomes by reducing the 2 M salt concentration of the mixture to 200 mM using a continuous rate dilution.⁵⁷
   1. Fill a syringe with 100 µL of dilution buffer containing 10 mM Tris pH 7.5 and place it on a syringe pump.
   2. Direct the needle of the syringe through a pre-punctured hole in the cap of the microfuge tube, ensuring contact is made with the assembly mixture (Figure 1).
   3. Run the syringe pump at a rate of 0.75 µL/min for 120 min.
      NOTE: The resulting 100 µL solution contains 250 nM nucleosomes and 200 mM NaCl.
   4. Transfer the mixture to a 10 K MWCO dialysis button and dialyze against 200 mL of a pre-chilled (4 °C) low salt buffer containing 10 mM Tris pH 7.5, 0.25 mM EDTA and 2.5 mM NaCl, for 1 hr at 4 °C.
6. To assess the histone content of the nucleosome assembly, prepare a discontinuous SDS-PAGE gel with a 15% separating and 6% stacking as previously described³⁰.
   1. Aliquot 10 – 20 µL of the nucleosome stock to a microfuge tube and add 4x Laemmli Sample Buffer to a working concentration of 1 – 2x.⁵⁸
   2. As a control, repeat this preparation in a separate microfuge tube for 1 – 2 µg of the histone stock. Heat the samples at 95 °C for ~ 5 min.
   3. Load the samples in adjacent lanes to one another on the gel. Add sample buffer to the unused lanes to promote even band migration.
   4. Run the gel at 65 V until the dye front moves through the stacking gel. When the separating gel is reached, increase to 150 V and run until the dye front has migrated completely out of the gel.
   5. Dismantle the electrophoresis unit and gently transfer the gel to a staining container filled with dd H₂O. Let the gel sit for 5 min with gentle agitation. Repeat this process twice more with fresh dd H₂O used each time.
      NOTE: The Coomassie stain used in this protocol does not require the typical fixing steps needed for Coomassie stains (see Table of Materials). If another Coomassie preparation is being used, adjust the fixing steps as needed.
   6. Remove the water from the final rinse and add just enough stain to cover the gel. Let the gel sit with gentle agitation for at least 1 h.
      NOTE: For the agitation, the staining container can be placed on any apparatus that promotes movement of the stain over the gel while also keeping the gel covered in liquid.
   7. Remove the stain from the container and rinse the gel with dd H₂O. Replace the dd H₂O and soak the gel for 30 min with gentle agitation. (The gel should appear like that shown in Figure 2, with clear separation of the histone bands.)
7. Store the nucleosomes at 4 °C until use.
   NOTE: When stored in these conditions, nucleosomes remain stable for several months. The protocol can be paused here.

2. Functionalization of Mica Surface for Static AFM Imaging of Nucleosomes

1. Prepare a 50 mM 1-(3-Aminopropyl) silatrane APS stock solution in deionized water as described.³⁰ Store 1 mL aliquots of this solution at 4 °C until use.
   NOTE: The aliquots can be stored for more than a year at 4 °C.⁵⁹
2. Prepare a working APS 1:300 solution for mica modification by dissolving 50 µL of the 50 mM APS stock in 15 mL dd H₂O.
   NOTE: This working solution can be stored at room temperature for several days.
3. Cut 1 x 3 cm strips of mica from high quality mica sheets (see Table of Materials for mica used here).
   1. Check that the piece fits when placed diagonally in a cuvette. Use the tip of sharp tweezers, a razor blade or scotch tape, to cleave layers of the mica until both sides are freshly cleaved and the piece is as thin as ~0.1 mm (Figure 3A). Immediately place the mica piece into the APS filled cuvette and incubate for 30 min (Figure 3B).
4. Transfer the mica piece to a cuvette filled with dd H₂O and soak for 30 s (Figure 3C). Completely dry both sides of the APS-mica strip under an argon flow.
   NOTE: A non-woven cellulose and polyester cleanroom wipe (recommended wipe detailed in materials) can be used to aid in wicking water from the edge of the mica when drying.
5. Use the dry mica strip is now for the sample preparation. Otherwise, store the piece in a clean, dry cuvette (Figure 3D).
   NOTE: Additional storage in a vacuum for 1-2 h is recommended when the environment is humid. The protocol can be paused here.

3. Preparation of Nucleosome Samples on APS-Mica for Static AFM Imaging

1. Apply double-faced adhesive tape to several magnetic pucks and place them to the side.
2. Cut the APS-mica substrate to the desired size (1 x 1 cm squares for the MM AFM instrument used here). Place these pieces in a clean petri dish and keep covered.
3. Prepare three dilutions of the assembled nucleosomes (final nucleosome concentrations of 0.5, 1.0 and 2.0 nM) using a 0.22 µm filtered buffer containing 10 mM HEPES pH 7.5 and 4 mM MgCl₂.
   NOTE: To limit the loss of nucleosomes at the low final concentration, the dilutions should be done one at a time, immediately prior to deposition on the APS-mica.
4. Deposit 5-10 µL of the diluted nucleosome sample at the center of the APS-mica piece, and let incubate for two minutes. Gently rinse the sample with 2-3 mL of dd H₂O to remove all buffer components. After each ~0.5 mL of dd H₂O used, gently shake the mica to remove the excess rinse water.
   NOTE: A disposable syringe is recommended for this rinsing step.
5. Dry the deposited sample under a light flow of clean argon gas.
   NOTE: The sample is now ready to be imaged or can be stored in a vacuum cabinet or desiccator filled with argon. Samples prepared and stored as described have been imaged one year following preparation with no quality loss. The protocol can be paused here.

4. Static AFM Imaging of Nucleosomes

1. Mount an AFM tip on the tip holder. Use a tip that has a spring constant of ~40 N/m and a resonance frequency between 300 and 340 kHz (see the Table of Materials for the cantilevers used here).
2. Mount the sample prepared in section 3 on the AFM stage being careful not to contact the sample surface.
3. Position the laser over the cantilever until the sum is at the maximum and adjust the vertical and lateral deflection values to near zero.
4. Tune the AFM probe to find its resonance frequency and adjust the drive amplitude and set the image size to 100 x 100 nm. Click the engage button to begin the approach.
5. Once approached, gradually optimize the Amplitude Setpoint until the surface of the sample is clearly seen. Increase the scan size to 1 x 1 µm and the resolution to 512 x 512 pixels. Click the capture button followed by the engage button to begin image acquisition.
   NOTE: The images in Figure 4 show the smooth background that can be expected when imaging these samples.
6. To analyze the nucleosome sample, open the captured images using the AFM instruments analysis software.
   1. Flatten the image using a polynomial line subtraction or similar feature.
   2. Set the color table to reflect the lowest value for the minimum and the highest value for maximum. Keep a record of these values for each image as they will be needed in a later step.
      NOTE: If these values are not used, height data will be incorrect in the later analysis as cross-sections of nucleosome cores will appear as plateaus of equal, rather than varying height.
   3. Export the image as a .tiff image at the original size. De-select any options to save the image with a border or scale bars.
   4. Open the image in analysis software capable of contour length measurements.
   5. Set the x, y and z scales to match the image.
   6. As an internal length calibration, measure and record the contour length of free DNA from one end to the other. For this calibration factor, use the measurements to generate a histogram and fit it with a normal (Gaussian) distribution. Divide the peak center (x_c) by the substrate length in base pairs.
      NOTE: This value is the image specific conversion factor from nanometers to base pairs of DNA.
   7. Measure and record the contour length of both arms for each nucleosome from the free end of the arm to the center of the core, for consistency (Figure 5A).
   8. For each nucleosome core, collect two full width at half maximum (FWHM) values from a perpendicular pair of core cross section measurements (Figure 5B Average the two FWHM measurements and subtract one-half of the resulting value from each nucleosome arm to correct for the measured length from the exit/entry DNA to the center of the core that is not part of the arm length.
   9. Divide each arm length by the calculated calibration factor (step 4.5.6) to obtain arm lengths in DNA base pairs.
   10. Calculate the extent of DNA wrapping by subtracting the nucleosome arm sum from the total base pair length of the unwrapped substrate. Plot these values as a histogram and fit the peak(s) with a normal (Gaussian) distribution to obtain the mean wrapped base pairs of DNA for the nucleosome population.
   11. Calculate the average nucleosome height from the measured cross sections. Plot this as a histogram and fit the peak(s) with a normal (Gaussian) distribution to obtain the height of the nucleosome population.

5. Time-Lapse AFM Imaging of Nucleosome Dynamics

1. Mount the AFM tip on the tip holder. A probe with a spring constant of approximately 0.1 N/m and a resonance frequency of 7-10 kHz can be used (see the materials list for the cantilevers used here).
2. Attach a 1 x 1 cm piece of APS-mica to a magnetic puck using double-stick tape and mount the puck on the AFM instrument.
3. Dilute the nucleosomes to a 1 nM concentration in a 0.22 µm filtered imaging buffer containing 10 mM HEPES pH 7.5 and 4 mM MgCl₂.
4. Deposit 5 – 10 µL of the diluted nucleosome at the center of the mica piece for 2 min. Rinse the deposited sample with 20µL of the imaging buffer two times. After the second rinse, keep a droplet of imaging buffer on the surface.
5. Use the top-view camera to find the tip and approach to the surface manually until the tip is ~100-500 µm from the surface.
6. Add additional imaging buffer to fill the gap between the tip and the surface. In this example, approximately 50 µL of imaging buffer is sufficient to fill the gap. Find a resonance peak for the tip.
7. Begin the computer-controlled approach to the surface. When approached, begin imaging with a 1-2 µm area to select an ~500 nm area of interest. With this area of interest selected, adjust the data acquisition density to 512 x 512 pixels.
8. Adjust the set point voltage and drive amplitude parameters to improve image quality. A free amplitude of 10 nm or less and a scan rate of ~2 Hz can be used to capture quality images. An example of nucleosome dynamics captured using time-lapse AFM is shown in Figure 6.

6. High-Speed Time-Lapse AFM Imaging of Nucleosome Dynamics

NOTE: The protocol below is provided for the HS-AFM instrument developed by the Ando group (Kanazawa University, Kanazawa, Japan).⁶⁰

1. Prepare APS-mica for liquid imaging.
   1. Attach the glass rod to the AFM scanner stage using the glass rod – scanner glue (see Table of Materials). Let this dry for a minimum of 10 min.
   2. Make ~0.1 mm thick circular pieces of mica with a 1.5 mm diameter by punching them from a larger mica sheet. Use the HS-AFM mica-glass rod glue (see Table of Materials) to attach this mica piece to the glass rod on the HS-AFM and dry, untouched for a minimum of 10 minutes. Cleave layers using a pressure-sensitive tape until a well cleaved layer is seen on the tape.
   3. Dilute 1 µL of 50 mM APS stock in 99 µL of dd H₂O to make a 500 µM APS solution. Deposit 2.5 µL of this solution on the freshly cleaved mica surface and let functionalize for 30 min.
      NOTE: To prevent drying of the surface while functionalizing, the cap of a 50 mL conical centrifuge tube can be fit with a damp piece of filter paper and placed over the scanner. The APS stock is diluted 3 times less for liquid imaging than for static imaging to control the dynamics to a rate that can be observed with AFM.
   4. Rinse the mica with 20 µL of dd H₂O by applying several ~3 µL rinses. Remove water completely following each rinse by placing a non-woven wipe at the edge of the mica. After the final rinse, place ~3 µL of dd H₂O on the surface and let it sit for a minimum of 5 min to remove any nonspecifically bound APS.
2. Place the probe in the HS-AFM holder and position the holder on the AFM stage with the tip facing up. Rinse the holder using ~100 µL of dd H₂O followed by two ~100 µL rinses of 0.22 µm filtered nucleosomes imaging buffer which contains 10 mM HEPES pH 7.5 and 4 mM MgCl₂.
3. With the rinses done, fill the chamber with ~100 µL of nucleosome imaging buffer, submerging the tip. Adjust the cantilever position until it is hit with the laser. Rinse the APS-mica with 20 µL of filtered nucleosome imaging buffer, using ~4 µL per rinse.
4. Dilute 1 µL of the nucleosome assembly stock into 250 µL of filtered nucleosome imaging buffer for a final nucleosome concentration of 1 nM. Deposit 2.5 µL of this dilution on the surface and let it sit for 2 min. Rinse the surface with ~ 4 µL of nucleosome imaging buffer two times. After the final rinse, leave the surface covered in imaging buffer.
   NOTE: If the surface is not rinsed after depositing the nucleosome sample, the surface will rapidly become overcrowded.
5. Set the scanner and sample on top of the tip holder so that the sample is face down. To begin the approach, use the auto-approach function with a set point amplitude, A_s close to the free oscillation amplitude A₀.
   NOTE: Ideally, A_s = 0.95 A₀, however, operating at 82% of A₀ will work as well if careful.
6. Adjust the set point until the surface is being well tracked.
   NOTE: To minimize the transfer of energy from the AFM tip to the nucleosome sample, the amplitude of the cantilever should be kept small, with amplitudes as low as 1 nm optimal.
7. Set the image area around 150 x 150 nm to 200 x 200 nm with data acquisition rate of ~300 ms per imaging frame.
   NOTE: This image size is typically sufficient to capture the dynamics of several nucleosomes simultaneously. A less populated surface may call for changes to these parameters. The suggested frame rate is sufficient to capture nucleosome dynamics such as looping, sliding and unwrapping, among others (see Representative Results section below).

7. Analysis of Nucleosome Dynamics Captured Using Time-Lapse AFM

1. Convert the images from the HS AFM data type (.asd) to .tiff images.
   1. Flatten the images using the AFM system’s analysis software using either plane or line functions until the background has uniform contrast.
   2. Set the image contrast (color scale) to automatic.
      NOTE: The contrast can be adjusted manually for presentation purposes but not for analysis. This causes height detail to be lost in the converted images.
   3. Save the selected range of images as a .mov file. Deselect the scale bar and border options before saving.
      NOTES: Do not do analysis on images that have scale bars in the frame. These alter the size of the image and will result in false measurements.
   4. Convert the .mov file to tiff images using a suitable software (see Table of Materials). Use the same frame rate for conversion as was used when creating the .mov file.
2. For arm length contour measurements, open the images in a measurement software capable of this measurement type (see Table of Materials).
   1. Set the image dimensions to match those at which it was captured.
   2. Measure the contour length of each nucleosome arm from the end of the arm to the center of the nucleosome core and record the measurements in a spreadsheet (Figure 4A).
   3. Measure the length of the unwrapped DNA substrate in the frames after a nucleosome unwraps. Use this for a movie specific calibration of the nm/bp ratio which is used for nucleosome wrapping calculations
   4. Collect two cross-section profiles for the nucleosome core and two for the bare DNA (Figure 4B).
   5. Import the cross-section profiles to a spreadsheet software and normalize the plots by subtracting the lowest z value from all points in the profile.
   6. Calculate the height and full width at half maximum (FWHM) for each cross section and average the values from the two profiles for each frame.
3. Subtract half of the FWHM value from each of the nucleosome arms are plot as a scatter plot along with the calculated sum of the arms.
   1. Calculate the mean contour length from the DNA measurements made for the DNA in frames after the nucleosome unwrapped.
   2. Determine the nm/bp calibration factor by dividing the mean contour length of the free DNA by the base pair length of the DNA substrate. Use this value to calculate nucleosome wrapping in each frame, as described in section 5.12.