Supramolecular Double Helices from Small C3-Symmetrical Molecules Aggregated in Water

Supramolecular fibers in water, micrometers long and several nanometers in width, are among the most studied nanostructures for biomedical applications. These supramolecular polymers are formed through a spontaneous self-assembly process of small amphiphilic molecules by specific secondary interactions. Although many compounds do not possess a stereocenter, recent studies suggest the (co)existence of helical structures, albeit in racemic form. Here, we disclose a series of supramolecular (co)polymers based on water-soluble benzene-1,3,5-tricarboxamides (BTAs) that form double helices, fibers that were long thought to be chains of single molecules stacked in one dimension (1D). Detailed cryogenic transmission electron microscopy (cryo-TEM) studies and subsequent three-dimensional-volume reconstructions unveiled helical repeats, ranging from 15 to 30 nm. Most remarkable, the pitch can be tuned through the composition of the copolymers, where two different monomers with the same core but different peripheries are mixed in various ratios. Like in lipid bilayers, the hydrophobic shielding in the aggregates of these disc-shaped molecules is proposed to be best obtained by dimer formation, promoting supramolecular double helices. It is anticipated that many of the supramolecular polymers in water will have a thermodynamic stable structure, such as a double helix, although small structural changes can yield single stacks as well. Hence, it is essential to perform detailed analyses prior to sketching a molecular picture of these 1D fibers.


Instrumentation and materials
Samples were vitrified using a computer-controlled vitrification robot (FEI Vitrobot TM Mark III, FEI Company). Quantifoil grids (R 2/2, Quantifoil Micro Tools GmbH) were surface plasma treated with a Cressington 208 carbon coater. Vitrified films were imaged using a Tecnai Sphera microscope equipped with an LaB6 filament operating at 200 kV and a bottom mounted 1024x1024 Gatan charged-coupled device (CCD) camera, or a CryoTITAN equipped with a field emission gun operating at 300 kV, a post-column Gatan energy filter for zero-loss filtering with a slit width of 20 eV, and a 2048 x 2048 Gatan CCD camera.
Samples for the image-based 3D reconstruction approach were prepared in Berlin by use of a Vitrobot TM Mark IV (Thermo Fisher Scientific). Quantifoil grids (R 1/4) were surface plasma treated with a BALTEC MED 020. Vitrified films were imaged with a Talos Arctica TM TEM (Thermo Fisher Scientific) at 200kV accelerating voltage employing a Volta phase plate in the back focal plane of the objective lens at a primary magnification of 48k. Images were recorded by a Falcon III direct electron detector at full 4k resolution resulting in a pixel size of 0.245 nm/pixel. The defocus value was set to -300 nm.

Sample preparation
Sample preparation of BTA polymers. Preparation of C12-MeBTA polymers: The solid C12-MeBTA was weighed into a glass vial and acetonitrile (ACN) was added to obtain a stock solution with a concentration of 25 mM. MilliQ water was added to a second glass vial and a small aliquot of the stock solution was added to obtain a sample with a BTA concentration of 500 μM (2% ACN). The mixture was stirred, using a magnetic stir bar, at 80 °C for 15 minutes. The hot and hazy mixture was subsequently vortexed for 15 seconds and was left to equilibrate at room temperature. Preparation of other BTA polymers: The desired amount of the compound was weighted into a sample vial and the respective volume of MilliQ water was added. Afterwards a stir bar was added, the mixture was vortexed for 10 s, then heated to 80 °C for 15 min while stirring and subsequently vortexed again for 10 s. The samples were left to equilibrate at room temperature overnight, before further experiments were performed.
Sample preparation of BTA copolymers. To prepare the solutions with copolymers, first, stock solutions of homopolymers were prepared according to the above described procedure. After cooling for 5 min, the solutions were mixed in the desired ratio, followed by 10 s of vortexing, heating to 80 °C for 15 min and subsequently vortexed again for 10 s. The samples were left to equilibrate at room temperature overnight, before further experiments were performed.
Vitrification of C12-nBTA without GOx. Samples of the C12-nBTA in MilliQ water were prepared at a concentration of 0.75 mg/mL using the procedure described above that was also previously reported for this compound. 1 The Vitrobot was operated at 22 °C and at a relative humidity of 100%. In the preparation chamber of the Vitrobot, a 3 μl sample was applied on a Quantifoil grid which was surface plasma treated for 40 s at 5 mA just prior to use. Excess sample was removed by blotting using two filter papers for 3 s at -3 mm, and the thin film thus formed was plunged (acceleration about 3 g) into liquid ethane just above its S3 freezing point. The vitrified film was transferred either to a cryoholder (Gatan 626) and observed in a Tecnai Sphera microscope, or to an autoloader cassette and observed in a CryoTITAN microscope, at temperatures below -170 °C. Micrographs were taken at low dose conditions.
Vitrification of C12-nBTA with GOx. GOx covered grids were prepared by depositing one drop of a vigorously stirred GOx solution at 0.1 mg/mL onto each grid. The grids were subsequently left to air-dry for about half an hour. Samples of the C12-nBTA in MilliQ water were prepared at a concentration of 0.75 mg/mL using the previously reported heating-cooling method. 1 5 nm gold particles in MilliQ water were added for tomography, resulting in a C12-nBTA concentration of 0.5 mg/mL. The Vitrobot was operated at 22 °C and prior to blotting and vitrification, the air in the preparation chamber of the Vitrobot was saturated with an isopropanol/water mixture (20/80 v/v), to reduce the high surface tension of the water film that may break during the extensive blotting that followed. The GOx coated grids were not surface plasma treated, because the GOx sheets are already hydrophilic. In the preparation chamber of the Vitrobot, 3 μl of the sample was applied to the grid. Excess sample was removed by blotting using two filter papers for 5 s at -3 mm, and using a drain time of 2 s. The thin film thus formed was plunged (acceleration about 3 g) into liquid ethane just above its freezing point. The vitrified film was transferred to an autoloader cassette and observed in a CryoTITAN microscope, at temperatures below -170 °C. Micrographs were taken at low dose conditions. Vitrification of other BTA (co)polymers. The samples for the image-based 3D reconstructions were prepared at a concentration of 500 µM as described above. The Vitrobot was operated at 22 °C and at a relative humidity of 100%. In the preparation chamber of the Vitrobot, a 5 μl sample was applied on a Quantifoil grid which was surface plasma treated for 60 s at 8.5 mA just prior to use. Excess sample was removed by blotting using two filter papers for 3.5 s at -3 mm, and using a drain time of 1 s. The thin film thus formed was plunged (acceleration about 3 g) into liquid ethane just above its freezing point. The vitrified films were transferred to the autoloader of a Talos Arctica microscope and imaged at low dose conditions.

Tomography and image reconstruction
Tomography. Tomograms were recorded using the CryoTITAN and using Inspect3D (FEI) software. Tilt series were recorded by tilting from -64° to 64° in 2° increments. Tracking correction was performed every tilt step. One tomogram was recorded of a vitrified film that contained multiple GOx sheets as support (multiple spots were observed in the diffraction pattern), at a magnification of 30000× and a nominal defocus of -2 µm. The exposure time per image was 0.2 s and the electron dose rate was 10 e / Å 2 s -1 . The second tomogram was recorded of a vitrified film on a GOx monolayer (only one set of spots in the diffraction pattern), at a magnification of 48000× and a nominal defocus of -1.5 µm. The exposure time per image was 0.5 s and the electron dose rate was 11 e / Å 2 s -1 . Alignment and reconstruction was performed using the IMOD software with the SIRT algorithm (volume size 951x1200x201, and pixel size 0.19 nm). 2 Subvolumes were extracted using the clip command prior to denoising, and to obtain numerical cross sections the Slicer module of IMOD was used. Filtering was performed on subvolumes in IMOD either using NAD with a K-value of 38440 and 100 iterations, or with a median filter of size 3x3x3.
Quantification in 2D and 3D. Measurements in 2D images were performed using Digital Micrograph. Median filtering in 2D was also performed using Digital Micrograph. To calculate the point resolution in 2D, the CTF was simulated using ctfExplorer v0.999a. For the Tecnai microscope operating at 200 kV, an energy spread of 1 eV, a focal spread of 4.72 nm, a convergence of 1.07 mrad, an object lens instability of 0.5 ppm and a spherical aberration coefficient (Cs) of 2 mm were used. For the CryoTITAN microscope operating at 300 kV, an energy spread of 0.6 eV, a focal spread of 4.01 nm, a convergence of 0.11 mrad, an object lens instability of 0.5 ppm and a Cs of 3 mm were used. FFTs of 2D images were obtained using Digital Micrograph and analysis of the distances in reciprocal space was performed using the Radial Profile plugin of Fiji. With this plugin a profile plot of normalized integrated intensities around concentric circles as a function of a distance from the origin was produced. The integrated intensity at any given distance from the origin represents the sum of the pixel values around a circle. This circle has the origin as its center and the distance from the origin as its radius, and the normalized values are then obtained by dividing the integrated intensities along the circle by the number of pixels in the circle. Measurements in 3D were performed using IMOD. Numerical cross sections with variable thickness were obtained using the Slicer module within IMOD. The same module was used to perform measurements in different perpendicular planes.

3D model generation and analysis from cryo-ET.
Manual segmentation of the supramolecular polymers was performed in the x-y planes (perpendicular to the beam direction) of the NAD filtered data using Avizo Standard 8.1.0. For both segmented polymers a LabelField was created and edited using the magic wand in the segmentation editor. Movie S2 of the segmentation results was prepared using Avizo's animation producer. To analyze the twist of the fibers in the x-z planes (y-direction), the LabelFields were exported as a .mat file to import the data in Matlab v2014b. Using a Matlab script, ellipses were fitted to the largest area of each cross-section along the y-direction. Since the segmentation was performed in the x-y planes, but the fitting of cross-sections in the x-z planes, these fits contain the two areas that are segmented (one for each BTA stack, see Figure S12). Subsequently the angles ('Orientation' in Matlab) of the long axis of the fitted ellipses were obtained. The angles were plotted as a function of the y-direction using Origin 2015. The eccentricities of the largest areas of the cross sections along the y-direction were 0.83 ± 0.11 and 0.81 ± 0.13 (where '0' is a circle and '1' is a line), hence the elliptical cross sections are a valid assumption.
2D image based helix reconstruction. The method of the image based helix 3D reconstruction scheme was reported previously. 3 For the case at hand 378 images of fiber segments (200x200 Pixel) were extracted from 20 different 2D cryo-TEM micrographs using the EMAN Boxer module. 4 All following operations were conducted by IMAGIC5 (Image Science GmbH, Berlin, Germany) image processing software. 5 The images were merged in a gallery file and subsequently band-pass-filtered to remove low-and high frequency noise. One single fiber motif was selected, rotationally and translationally centered and used as reference for a first round of fiber alignments. The sum of aligned images with a correlation factor better than 0.4 compared to the reference was used as a new reference. This alignment procedure was repeated until correlation factors were stable. Multivariate statistic (MSA) was applied to the aligned data set followed by classification. MSA is a method based on correspondence analysis 6 and was introduced in image processing procedures of large electron microscopic data sets of "single particles" (e.g. a population of biomacromolecules) to determine subsets of these particles according to their distinct structure-related features (e.g. different spatial orientations) without introducing subjective criteria. 7 Subsequent classification (= subsets of images) revealed deviations in the data set such as bending, poorly aligned images or distortions in the supramolecular organization. The class sum with the smallest intra class variance was used for the helical 3D volume reconstruction. A full pitch repeat motif (known from the cryo-ET volume data) was extracted from the class sum image and blown-up so that the motif becomes 360 pixels long. This image was cyclically shifted in y-direction in single pixel steps which is the equivalent of a 1° rotation of the fiber motif. The corresponding Euler angles were assigned to the 360 shift images and the reconstruction was calculated using the IMAGIC "true 3D" weighted back projection algorithm and is shown in Figure 3c as surface representation. The determination of all other presented class sum images shown in Figures 3 and 4 followed principally the same above described procedure.   . Low-dose electron diffraction (LDED) pattern that was recorded after the tomographic series was acquired, for the tomographic series that was studied in detail as described in the manuscript. The image shows a single set of diffraction spots of the GOx support layer (scale bar = 1 nm -1 ). Figure S4. Numerical cross-section (average over six slices, 1.1 nm) that is parallel to the air-water interface and a four-times magnification in the inset (scale bar = 20 nm). Figure S5. Cartoon representation to show the hypothesized edge-on orientation of the C12-nBTA molecules at the air-water interface using a side-view of the vitrified water film. The stack of molecules on the interface shows the hydrophobic core in gray, the amides in lightgray, and the protruding side arms have a gray part for the aliphatic C12-chains and a blue part for the ethylene glycol oligomers. Figure S6. Result from a second tomographic tilt series, that was recorded at a lower magnification (30000× instead of 48000×). The alignment and reconstruction in IMOD were performed using the same procedure. The top image is a numerical cross-section through the reconstruction (average over ten slices, 2.8 nm) of a 3D SIRT (simultaneous iterative reconstruction technique) reconstruction of a tomographic series recorded using a vitrified film on multiple layers of GOx. The data was filtered in three dimensions using a median filter of size 3x3 before summing along the z-direction (beam direction). Along with the supramolecular polymers, smaller aggregates were observed as indicated by the black circles. Due to limitations in contrast and resolution these small aggregates were never observed before in conventional cryo-TEM imaging. The high contrast straight lines at the right bottom and top S10 left are the edges of the GOx sheets used to suspend the samples (indicated with white arrows), and the grey particles are 5 nm gold particles added for alignment of the tilt series. The dimensions and shapes of the small aggregates were analyzed by extracting numerical vertical cross-sections (average over ten slices, 2.8 nm) in the y-direction (x-z planes; a=b, d=e) and xdirection (y-z planes, c and f) (bottom image). The numbers refer to the distance between the yellow and red crosses in the images. The small aggregates have an irregular shape and are typically less than 3 nm in diameter. Figure S7. A) Numerical cross-section (average over 50 slices, 9.5 nm) through the 3D SIRT reconstruction of the tomographic series recorded at a magnification of 48000×, that was filtered in three dimensions using NAD with a K-value of 38440 and 100 iterations before summing along the z-direction. The dashed box indicates the region in which the variations in contrast can be observed most clearly. B) Numerical cross-section equal to A), but with a red line that indicates the x-position along which the numerical cross-section in the y-z plane was obtained (in C). C) This numerical cross-section (average over 32 slices in the x-direction, 6.1 nm) indicates that all contrast that originates from the part of the polymer that is in between the white lines (that have the same y-position in images B and C) is included in the crosssection (A, B). The C12-nBTA concentration is 0.5 mg/mL in H2O.

Figure S8.
To allow a fair comparison of the 2D diameters of Figure S2 with the data obtained using Cryo-ET, all z-slices in which (fractions of) the polymers were observed were averaged to yield the image as displayed. Specifically, it is a numerical cross-section (average over 50 slices, 9.5 nm) obtained by summing along the z-direction (beam direction). The data was filtered in three dimensions using a median filter of size 3x3. The white numbers originate from measuring the smallest diameter of the supramolecular polymers, and the black numbers correspond to the largest diameters. The numbers with an increased font size at the bottom-left are the average and standard deviation, with 'n' the number of measurements. The grey particles are 5 nm gold particles added for alignment of the tilt series.  Figure 2 of the main text, originating from the NAD (left) and median filtered data (middle and right). The measurements in white are the diameters of the single stacks of molecules, and those in black correspond to the width of the double helix. Figure S10. The angles of the long axis of the ellipses that were fitted to the largest segmented areas (that can consist of two areas where two separate stacks are observed, see Figure S12), as a function of the position along the polymers in the y-direction. Both graphs were constructed from the parts of the polymers that were oriented parallel to the y-axis, and graph A) was partially used for Figure 2E. In graph B) some fluctuations are observed due to segmented cross sections that were circular in shape. The periods 1 -8 as indicated in A) and B) were used to calculate the average length of a period (22.9 nm, s.d. 1.8 nm), because these parts were most straight (parallel to the y-axis) as observed in the 3D models and without many circular cross-sections. Figure S11. Cryo-TEM image of supramolecular polymers formed from C12-nBTA (0.75 mg/mL in H2O), in a vitrified film without using GOx. The imaging was performed at a magnification of 61000× and -5 µm defocus, and subsequently a median filter of size three was applied. The white numbers originate from measuring the periods of the twist of the supramolecular polymers, and the numbers with an increased font size at the bottom-right are the average and standard deviation, with 'n' the number of measurements. Figure S12. The top row shows cross-sections of the NAD filtered data (x-z planes) with in magenta an overlay of the segmented areas that clearly consist of at least two different areas. The cross-sections were obtained in the y-direction at the following positions (that correspond to those in Figure 2E of the main text): 109 nm (left), 145 nm (middle) and 169 nm (right). The bottom row shows numerical cross-sections at the same positions along the y-direction (averages over ten slices, 1.9 nm), with yellow lines of which the length corresponds to the calculated length of the long axis of the ellipses that were fitted to the segmented areas as indicated in the top row, showing that the occurrence of two separate areas is taken into account. Similarly, the orientation of the long axis was set to match with the calculated angle.

Synthesis of the new compounds
Synthesis of C16-dBTA. All reagents and solvents were obtained from commercial suppliers and used without further purification unless stated otherwise. pG1-OH was synthesized according to published procedures. 8 Reactions requiring dry and oxygen-free conditions were carried out in oven-dried glassware with septa using usual Schlenk techniques. Millipore water (Milli-Q) was obtained from a Merck Millipore Milli-Q Integral System. NMR spectra were recorded on JEOL ECX400, JEOL ECP500, BRUKER AV500 and BRUKER AV700 spectrometers at 400 MHz, 500MHz and 700MHz for 1 H NMR spectra and 100 MHz, 125MHz and 175MHz for 13 C NMR spectra, respectively. Chemical shifts are given in parts per million (ppm) in relation to deuterated solvent peak calibration. Flash columns were either performed with Macherey-Nagel silica gel 60M or at a Combiflash Rf system from Teledyne ISCO.  Figure S17. Synthesis scheme of C16-dBTA.

NH2-C16-pG1 -(4)
Phth-C16-G1 (3) (0.43 g, 0.62 mmol) was dissolved in 60 mL ethanol. Hydrazine monohydride (0.75 mL, 15.50 mmol) was added and the reaction mixture was heated to 80 °C and kept stirring under reflux overnight. Subsequently, the solvent was removed under reduced pressure and the residue was redissolved in NaOH (1M) and extracted three times with DCM. The combined organic phases were dried with Na2SO4 and the solvent was removed under reduced pressure. The title compound (0.34 g, 98%) was obtained and used without further purification in subsequent reactions.