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Nature Methods Recovery of intact DNA nanostructures after agarose gel–based separation Gaëtan Bellot, Mark A McClintock, Chenxiang Lin & William M Shih
Supplementary Figure 1
Electroelution of DNA nanostructures from agarose into sucrose bed.
Supplementary Figure 2 Agarose-gel analysis of DNA-origami after varied glycerol concentration gel purifications.
Supplementary Figure 3 Agarose-gel analysis of DNA-origami after varied sucrose concentration gel purifications.
Supplementary Figure 4
Supplementary Figure 5 Agarose-gel analysis of DNA-origami purified by ion-exchange column and sucrose gel purification.
Supplementary Figure 6
Supplementary Figure 7 Characterization of DNA-nanotubes liquid crystal.
Supplementary Figure 8 Gel-shift analysis of the tetramerization of twelve-helix bundle.
Supplementary Figure 9 Transmission electron microscopy of tensegrity structure after gel purification.
Supplementary Table 1 Folding of DNA-origami shapes.
Supplementary Methods
Nature Methods vol. 8, no. 3 Shih, W.M. et al.
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Nature Methods vol. 8, no. 3 Shih, W.M. et al.
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Nature Methods vol. 8, no. 3 Shih, W.M. et al.
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SUPPLEMENTARY TABLE 1
DNA-origami shapes
Scaffolds from M13mp18 Folding buffers
Thermal annealing ramp
Six-helix bundle p73085 mM Tris-HCl pH 8 at
20°C, 1 mM EDTA, 18 mM MgCl2, 5 mM NaCl
80˚C to 60˚C in 80 min, 60˚C to 24˚C
over 18 hours
Twelve-helix bundle p80645 mM Tris-HCl pH 8 at
20°C, 1 mM EDTA, 12 mM MgCl2, 5 mM NaCl
80˚C to 60˚C in 80 min, 60˚C to 24˚C
over 24 hours
Six helix-bundle DNA ring
p30245 mM Tris-HCl pH 8 at
20°C, 1mM EDTA, 10 mM MgCl2,
80˚C to 60˚C in 80 min, 60˚C to 24˚C
over 72 hours
“Tensegrity” structures
p86345 mM Tris-HCl pH 8 at
20°C, 1 mM EDTA, 16 mM MgCl2
80˚C to 60˚C in 80 min, 60˚C to 24˚C
over 72 hours
Supplementary Table 1 | Folding of DNA-origami shapes.
Nature Methods vol. 8, no. 3 Shih, W.M. et al.
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SUPPLEMENTARY METHODS
Reagents and equipments. 2xYT Microbial Medium (Sigma). Magnesium chloride, polyethylene glycol 8000 (PEG8000), sodium chloride, Tris base, sodium hydroxide, potassium acetate, lauryl sulfate, glacial acetic acid (Fisher Scientific). 8-well PCR strip tubes (Molecular BioProducts). Agarose (Invitrogen).Sucrose, glucose (Sigma).Freeze ‘N Squeeze DNA gel-extraction spin columns (Bio-Rad). Pellet pestles (Kimble-Chase). Carbon/formvar copper grids (SPI). RPC-purified deoxyribonucleotides (Bioneer). Thermal cycler (MJ Research). Gel box 12x14 cm, OWL Easycast B2 apparatus (Thermo Scientific). UV lamp (Entela).
Folding of the DNA-origami shapes. Assembly of the DNA nanostructures is accomplished in a one-pot reaction by mixing scaffold strands derived from M13 bacteriophage with five times excess of every oligonucleotide staple strands (reverse-phase cartridge purified, Bioneer Inc.) in a folding buffer and subjecting the mixture to a thermal-annealing ramp that cooled from C to
over the course of 80 minutes and then cooled from C to C over 18 to 72 hours depending of the shape. The details of the folding reaction for each DNA nanostructure can be found as Supplementary Table 1 online and see Supplementary Figures 7–8 for detailed examples of how staple strands can be programmed to link the scaffold strand into a twelve-helix bundle and six-helix bundle ring structure. Nanotube oligomer were formed by combining front
Agarose gel analysis. The folded objects were electrophoresed on a 1.5% agarose gel containing 0.5x TBE buffer (45 mM Tris boric acid, 45 mM Tris base, 0.5 g/mL ethidium bromide, 11 mM MgCl2 and 1 mM EDTA (pH8) ) at 60 V for 3 h. To protect the Prestressed DNA tensegrity structures Kites from denaturation during electrophoresis, the gel-box was cooled in an ice-water bath.
Gel Purification Procedure.Gel Casting1. Thin and rigid bottom layer : pour 50 mL 4% agarose in 0.5x TBE + 11 mM MgCl2 into casting tray, let solidify.
2. Resolving layer : pour 120 mL 1% agarose in 0.5x TBE, 11 mM MgCl2, 0.5µg/mL ethidium bromide, on top of 4% agarose layer.
3. Add in comb. Elevate the comb so that there is a few mm separation between the bottom of the comb and the 4% bottom agarose layer. Let solidify.
4. Submerge gel in electrophoresis chamber with 0.5xTBE + 11 mM MgCl2, electrophorese at 60V for 2 hours at room temperature or higher voltage in an ice-water bath.
Gel purification 1. Remove the gel from the electrophoresis chamber and empty buffer. Cut out well in front of or/and behind band of interest. Do not to cut into 4% agarose layer. It’s possible to cut well behind band to get separation from multimers;; thus cutting this well is optional.
2. Return tray to chamber, add 0.5xTBE + 11 mM MgCl2 to the chamber up to the level of the gel. Do not add buffer above this level;; the surface of the gel should still be exposed.
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3. Wash the well with the Electroelution Buffer (30% sucrose, 11 mM MgCl2, 0.5xTBE) and fill the wells with the same solution.
4. Electrophorese the band into the sucrose bed. Recover the band with a pipettor.5. Desalt by serial dilution of buffer in microcon spin column, or other method of choice.
Pellet-pestle homogenization gel purification. Folding products were electrophoresed on 1% agarose gel containing 0.5x TBE, 11 mM MgCl2, 0.5 !g/ml ethidium bromide at 75 V for two hours in a gel box incubated in an ice-water bath. Bands of interest were excised and DNA recovered by pestle-crushing excised bands followed by centrifugation for 10 min at 16000 rcf at 4ºC using Freeze ‘N Squeeze DNA Gel Extraction spin columns (Bio-Rad). Recovered material in the flow-through was stored at 4ºC for further use.
Ion-exchange column purification. Buffer QBT : 50mM MOPS pH 7.0, 750mM NaCl, 15%(v/v) Isopropanol, 0.15%(v/v) Triton X-100. Buffer QC : 50mM MOPS pH 7.0, 1M NaCl, 15%(v/v) Isopropanol. Buffer QF : 50mM Tris pH 8.5, 1.25M NaCl, 15%(v/v) Isopropanol.Equilibrate the Qiagen-Tip 100 column in 4mL Buffer QBT. Allow to flow through completely. Apply 5mL sample to column, and allow to flow through completely. Wash the column 4x with 10mL Buffer QC allowing the buffer to flow through the resin completely for each wash. Elute the DNA with 5mL Buffer QF.
Gel-based yield estimation. ImageJ (http://rsb.info.nih.gov/ij/) was used for gel-image analysis to estimate the yield of purification. Standard deviation reported was done on six different purifications. Agarose-gels were calibrated (agarose gel concentration, buffer, gel dimension, gel thickness, ethidium bromide staining) to provide the highest precision in the quantification process. The percentage of recovery that partitioned as a monomeric species was estimated as the background-subtracted integrated intensity value of a selection box enclosing the band of “purified” lane divided by the background-subtracted integrated intensity value of a selection box enclosing the “unpurified” lane material band.
Transmission electron microscopy. For imaging, particles were adsorbed onto glow discharged collodion and carbon coated TEM grids and then stained using a 2% aqueous uranyl formate solution containing 25 mM NaOH. Imaging was performed using an FEI Tecnai T12 BioTWIN operated at 80 kV. Negative stain electron microscopy purified samples were adsorbed for 5 min onto glow discharged formvar-and carbon coated copper grids, stained for 1 min with 2% uranyl formate, 25 mM NaOH, and visualized at 68000x magnification with an FEI Tecnai T12 BioTWIN operating at 80 kV.
Nature Methods vol. 8, no. 3 Shih, W.M. et al.
