Biomod/2013/Dresden/labbook

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               <li><a href="http://openwetware.org/images/4/4e/Hydrophobic_Modifications_Protocols.pdf">Hydrophobic_Modifications_Protocols.pdf</a></li>
               <li><a href="http://openwetware.org/images/4/4e/Hydrophobic_Modifications_Protocols.pdf">Hydrophobic_Modifications_Protocols.pdf</a></li>
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               <li><a href="openwetware.org/images/4/42/Origami_Folding_Protocols.pdf">Origami_Folding_Protocols.pdf</a></li>
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<li><a href="openwetware.org/images/6/60/Protocols_for_Internal_Reaction.pdf">Protocols_for_Internal_Reaction.pdf</a></li>
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<li><a href="http://openwetware.org/images/6/60/Protocols_for_Internal_Reaction.pdf">Protocols_for_Internal_Reaction.pdf</a></li>
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               <li><a href="openwetware.org/images/0/06/Protocols_for_Polymersome.pdf">Protocols_for_Polymersome.pdf</a></li>
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               <li><a href="http://openwetware.org/images/0/06/Protocols_for_Polymersome.pdf">Protocols_for_Polymersome.pdf</a></li>
<li><a href="openwetware.org/images/2/27/Staples_sequence.pdf">Staples_sequence.pdf</a></li>
<li><a href="openwetware.org/images/2/27/Staples_sequence.pdf">Staples_sequence.pdf</a></li>
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Revision as of 13:58, 26 October 2013

Protocols

Polymersome protocols

Triblock polymersome

For the synthesis of the triblock polymersome we followed the protocol described in Corinne Nardin et.al [1]. In brief, the following steps were carried out:

  1. 5ml pear shaped flask was washed with pure ethanol and dried at 120°C for 1h.
  2. 1 mg polymer was dissolved in 1mL ethanol.
  3. Solution was evaporated at 40°C (water bath temperature), 40 rpm (rotation speed at an angle as high as possible, ~ 60°-70°) and a pressure of 150 mbar in rotary evaporator.
  4. After complete evaporation of ethanol, pressure was reduced using rotary pump vacuum (~10-2 mbar) for 30 min.
  5. Later 1mL PBS (pH 7.4) was added and stirred overnight at 300rpm.
  6. In order to remove residual lamellar material and to define particle size less than 200nm, samples were extruded using ultra-filtration equipment (Avanti Polar miniExtruder) with Nucleopore membrane (0.2µm pore size).

Diblock polymersome

For the synthesis of the diblock polymersome, we followed as described in Mohamed A. Yassin, et.al [2], in brief, the following steps were carried out.

  1. Millipore water was used for solution preparation.
  2. 1mg Polymer was dissolved in HCl (pH 2) with the help of magnetic stirrer at 700rpm.
  3. Later the pH of solution was reduced from 2 to 8, by adding NaOH drop wise at very slow rate. The pH of the solution should be checked very carefully.

DNA origami protocols

Preparation of staple mixtures and folding buffers

The 5 types of staples: core (118 staples), edge (39 staples), no hang (12 staples), helper (12 staples) and anchor (12 staples) were ordered in Biomers, already purified by reverse-phase cartridge.

  1. 194 staple sample tubes were dissolved with MilliQ water according to information provided by the company. (ensured that the concentration is 100 µM, but need Nanodrop to see if the concentration is right).
  2. Five mixtures of staple types were made: for each type, 5µL of each sample were added and mixed well by vortexing.
  3. Dilutions of core, edge, no hang, anchor and helper staples to the final concentration of 500, 2000, 4000, 4000 µM respectively were made by adding appropriate amount of MilliQ water.
  4. Folding buffers containing different concentrations of Magnesium Chloride were prepared: 80, 100, 120, 140, 160, 180 and 200 mM from 1M magnesium chloride stock in 10X TE buffer (50mM Tris, 10mM EDTA).

Folding reaction

The mixture of scaffolds, staples and folding buffer (FB) are prepared in a total volume of 100 µl.

Concentration (nM)Volume (µl)Final concentration (nM)
Scaffold1001515
core50020100
edge20005100
No-hang/anchor40002.5100
helper40002.5100
10X FB101x
Milli Q H2O45

For the incorporation of the hydrophobically modified oligos, a final concentration of 400 nm was used, and their incorporation was tried during the thermal annealing ramp, and once it was already assembled.

The mixture was subjected to a thermal annealing ramp, following the schedule adapted from “A logic gated nanorobot for targeted transport of molecular payloads” by Douglas et al. Science 2012:

  • 80°C to 61°C at 2 min/°C
  • 60°C to 24°C at 60 min/°C

Filtration

Amicon ultra 0,5 mL filters of 100 KDa cut-off were used, from Millipore.

  1. Filter was placed to the special eppendorf and the sample was transferred (100 µL).
  2. Another 200 µL of folding buffer 1x were added.
  3. Centrifugation 10’ 6000 rpm at table centrifuge.
  4. 300 µL folding buffer 1X were added and centrifugation again 10’ 6000 rpm.
  5. The lower eppendorf was discarded, and the filter was placed to a new one, but this time the filter inverted.
  6. Centrifugation 7’ 7000 rpm.
  7. Transference to a new tube.
The protocol was provided by Bezuayehu Teshome, Helmholtz-Zentrum Dresden Rossendorf.

Short electrophoresis

Gel preparation

  1. An agarose 1% solution was prepared with TAE 1X buffer.
  2. The solution was heated up until boiling and let cool down until 55ºC
  3. A magnesium chloride solution was added to reach a concentration of 12mM.

Sample loading

Samples were mixed with the orange G-based loading dye in a ratio 1:5.

Gel running and staining

Gels were runned for 1h 30’ at 60V, and then stained with Ethidium bromide for 20’ for further UV visualization.

5' Palmitate and arachidic acid modification

To attach the hydrophobic modifications to the oligonucleotides, those were ordered with a 5’ amino group that reacts with the carboxylic group present in the molecule that contains the hydrophobic groups. This is an N-acylation reaction, and its general mechanism is depicted in the following figure. The reaction is not spontaneous, and so the activators HATU and HOAt are required.

N-acylation reaction

Oligonucleotides still attached to the CPG beads were ordered from Biomers, with the 5’-amino-C6-modifier and the Monomethoxytrityl (MMT) group still on. The nucleotide sequence was the following: 5'-CGCGGATGGCGATGCGCGCAC-3'.

The amino-C6-modifier confers the amino group necessary for the attachment of the hydrophobic modifications.

N-acylation reaction

The MMT group was then removed and the Fmoc-L-dap(Palmitate)-OH molecule (Cas nº 724785-41-5) was introduced, carrying the palmitic acid, and the carboxylic group that will react with the amino group present in the oligonucleotide. With a similar reaction, the amino group from this molecule was deprotected from the Fmoc molecule, and the arachidic acid (Cas nº 506309) was introduced in this position.

The steps were the following:

  1. Weighing of reagents and dissolving in DMF.
    • HATU 6.8 mg in 40µl DMF.
    • HOAT 2.18 mg in 20µl DMF.
    • Fmoc-L-DAP(Palm)-OH 12.3 mg in 100µl DMF.
  2. Let dissolve in Ultrasonic bath for 5’
  3. First mix HATU + HOAT, then add Fmoc-L-dap(Palm)-OH. Incubate 1h RT under agitation (meanwhile steps 4-6)
  4. Wash beads with 1mL Deblock (dissolved in DCM) to remove MMT group. Trityl is yellowish, repeat until solution is clear.
  5. Wash beads 3x with DCM.
  6. Let beads dry, if necessary using vacuum centrifuge.
  7. After the 1h reaction, add the mixture to the beads + 60µl TEA 1M. Let react for 5h or ON at 30ºC.
  8. Wash beads with DMF until the solution appears clear.
  9. Wash 3x with DCM, let dry or vacuum dry.
  10. Weighing of reagents to couple arachidic acid:
    • HATU 6.8 mg in 40µl DMF.
    • HOAT 2.18 mg in 20µl DMF.
    • Arachidic acid 6.25 mg in 100µl DMF.
  11. Let dissolve in Ultrasonic bath for 5’.
  12. First mix HATU + HOAT, then add arachidic acid. Incubate 1h RT under agitation (meanwhile steps 13-14)
  13. Wash the beads 3x with diperidine for the Fmoc decouping.
  14. Wash 3x with DCM, let dry or vacuum dry.
  15. Add reagent mixture + 60µl TEA.
  16. Let react ON at 30ºC under agitation.
  17. Wash beads 3x with DMF, 3x with DCM and let dry or vacuum dry.
To release the oligos from the beads and also remove the protective groups from the bases ammonia was added and DNA was let dissolve while beads precipitate. Then ammonia was evaporated in vacuum and the modified oligos were solubilized with water. The modified oligo was purified by HPLC and its purity was analyzed my mass spectrometry.

Protocol provided by Francesco Reddavide, currently working in Yixin Zhang's lab, B-CUBE, Dresden.

3' Cholesterol modification

For cholesterol modification, the oligonucleotides were synthesized on a solid support where cholesterol was previously attached. The sequence was the following: 5'-CGCGGATGGCGATA-3'.

In this case it is shorter for the modification to be slightly separated from the walls of the channel. To release the oligos from the beads and also remove the protective groups from the bases ammonia was added and DNA was let dissolve while beads precipitate. Then ammonia was evaporated in vacuum and the modified oligos were solubilized with water. The modified oligo was purified by HPLC and its purity was analyzed my mass spectrometry.

Protocol provided by Francesco Reddavide, currently working in Yixin Zhang's lab, B-CUBE, Dresden.

Long electrophoresis

Gel preparation

  1. An agarose 1% gel was prepared in a TBE 0.5X solution.
  2. The solution was heated up until boiling and let cool down until 55ºC.
  3. A magnesium chloride 1M solution was added to reach a concentration of 12mM.

Sample preparation

  1. The samples were diluted with folding buffer with a concentration of 12mM MgCl2 and 100ng of DNA were loaded in each well.
  2. Samples were mixed with an orange G-based loading dye in a ration 1:5.

Gel running and staining

Gel was runned with a running buffer consisting of TBE 0,5X with 12mM MgCl2, at 70V during 4 hours. The electrophoresis cell was maintained surrounded by ice for the system not to heat up, and the buffer was changed and the electrodes cleaned after 2h. The gel was then stained with Ethidium bromide during 20’ for the further visualization under UV light.

Protocol provided by Alexander Ohmann, currently working in Stefan Diez’s lab, in B-CUBE, Dresden.

Internal reaction protocols

Conjugation of TAMRA to collagenase

The TAMRA amine-reactive fluorescent dye (5-(and-6)-carboxytetramethylrhodamine, succinimidyl ester (5(6)-TAMRA, SE)) from Invitrogen was conjugated to the Collagenase from Clostridium hystoliticum from Sigma, Type V.

The labelling protocol was the following:

  1. 10 mg of enzyme are dissolved in 1 ml of 0.1M sodium bicarbonate buffer, pH 8.3.
  2. 5 mg of dye are dissolved in 500 µl of DMSO. The dye should be dissolved immediately before the reaction. The solution is protected from light.
  3. While vortexing the protein solution, 100 µl of the TAMRA solution is added slowly. This corresponds to 1 mg of dye into 10 mg of protein. TAMRA NHS ester is amine-reactive dye and reacts with non-protonated aliphatic amine groups, including the amine terminus of proteins and the є-amino group of lysines.
  4. The reaction is incubated for 1h at room temperature with continuous stirring and protected from light.
  5. Separating the conjugate from the unreacted dye is performed using Sephadex G-25 size exclusion column. The first excluded fraction which corresponds to the first fluorescent band to elute is the conjugate.
  6. The protein concentration is determined after collecting the fractions. The protein-dye conjugate is diluted 100 times. The absorbance of the diluted sample is measure at 280 nm and at 555 (the excitation maximum).
The protein concentration is determined using the following formula:

Aprotein-A<555>(CF)

CF is the correction factor used to correct for the contribution of the dye to the absorbance at 280nm.

The protein concentration is calculated assuming that 1.7 Aprotein units = 1mg/ml. This value is different for each protein and it was experimentally determined for collagenase.

The degree of labeling is calculated using this formula:

D.O.L. = (Amax x MW)/([protein])x Edye

MW = molecular weight of the protein, Edye= excitation coefficient of the dye at its absorbance maximum, [protein] = protein concentration in mg/ml.

Nanoreactor protocols

Release profile assay

The release profile assay was performed using cellulose dialysis membrane of MWCO 5000 Da and 300 KDa for biological entities less than 500 Da and Enzymes respectively. Procedure:

  1. The beaker was filled with dialysis solution (Milli-Q water or Buffer solution).
  2. Dialysis tube was cut as per the volume of sample and dialysis tube width.
  3. The tube was washed in Milli-Q water and pre-incubated in Milli-Q water for 20 min.
  4. After pre-incubation, the tube was closed on 1 end with magnetic clamp such that atleast 10 mm tubing overlaps.
  5. Then through the other end, sample was filled inside the tube and air was removed carefully before closing the tube with clamp as before.
  6. Dialysis tube was placed in prepared beaker and stirred at low speed (300 rpm).
  7. Dialysis solution was replaced in beaker after every 2 hours.
  8. After each hour, samples were analysed using Beckman Coulter – UV 800 Spectrophotometer for Doxorubicin, enzyme, Tamra dye, fluorescein dye at 480, 280, 555 and 490 nm respectively. After dialysis, the sample was removed carefully after drying the tube with kimwipe.

Incorporation and encapsulation of biological entities

Incorporation of DNA channel (15nM conc.) and Encapsulation of enzymes (20% of polymer conc.) was carried out by adding during each polymersome preparation in following ways.

  1. PMOXA-PDMS-PMOXA: DNA channel or Enzyme was added to PBS buffer in laststep along with 5µl MgCl2 solution (1 M).
  2. (PEG-b-P(DEAEMA-stat-BMA)): Polymersome solution pH was brought to 5 and DNA channel (15nM conc.) or Enzyme was added along with 5µl MgCl2 solution (1 M).Further, solution pH was reduced further to 8.

Enzymatic assay with nanoreactors

The final reaction assay with nanoreactors were performed using the tri-block and di-block polymersomes without channels as a negative control and the polymersomes with channels with different hydrophobic modifications. The reaction assay was performed for lower and higher concentration of the polymersomes - 22 µg/ml and 100 µg/ml respectively. The reaction mixtures were incubated for the same time and samples were taken for 3h, 6h, 8h 30 min, 24 h and 48h. These samples were checked with UPLC for the presence of reaction products.

Imaging and analysis

Cryo-TEM (Transmission Electron Microscope)

  1. For frozen hydrated specimen preparation, 2µl of sample was deposited on a holey carbon support film attached to a copper grid.
  2. The liquid film was thinned by blotting for 0.5 sec and frozen rapidly in liquid ethane.
  3. After rapid freezing, the sample was loaded to a Gatan cryo transfer holder keeping temperature below -130°C in order to prevent ice crystallization.
  4. TEM inspection was done in ZEISS LIBRA 120, energy filtered images were recorded under low dose electron irradiation conditions.
  5. Samples were kept below -130°C during the inspection.

Cryo-SEM (Scanning Electron Microscope)

  1. Samples were rapidly frozen in liquified ethane according to standard procedure.
  2. Frozen samples were fractured at approximately -130°C in a cryo preparation chamber and transferred to the cooling stage inside a ZEISS Ultra after coating with 3nm Carbon layer.

AFM (Atomic Force Microscopy)

Sample preparation

A negatively charged mica substrate was used to absorb the DNA origami samples.

  1. The substrate was cleaned with isopropanol and a scotch tape to remove the impurities.
  2. The mica layer was rinsed with MilliQ H2O, and immediately dried with a nitrogen gun.
  3. 2 µl of the DNA origami sample at a concentration of about 0,5 nM were placed on the substrate and incubated for 2-5’.
  4. The substrate was washed with Milli Q H2O to get rid of the salts.
The protocol was provided by Bezuayehu Teshome, Helmholtz-Zentrum Dresden Rossendorf.

Device characteristics

AFM Probe Type: Tap150AI-G, from BudgedSensors. Rotated tip shape, height of 15-19 µm and radius of < 10 nm.

TEM (Transmission Electron microscopy)

Grid preparation

Grids were prepared by Susanne Kretschmar, working in the EM facility in CRTD.

  1. Plasma cleaning to hydrophilize the surface.
  2. Grids were placed on a glass slide wrapped in parafilm, and freshly glow discharged for 60s at 240V.

Sample preparation and staining

A DNA origami sample concentration of approx. 5 mM was prepared.

  1. 5 µl of the samples were place on the grid and incubated for 2’. The excess liquid was dried with a filter paper.
  2. A dilution of uranyl acetate 2% in H2O was centrifugued at 14500g during 3’ to precipitate the big granules.
  3. A drop of this solution was placed on the samples and incubated for 20’’, then it was removed with a filter paper.
  4. A water drop was placed to clean the excess and removed with a filter paper.
  5. The grid was allowed to air-dry before imaging.

Device

The microscope used was Morgagni 268 at 80kV

CLSM (Confocal Laser Scanning Microscopy)

The confocal images were taken using Zeiss LSM 780/FCS confocal microscope. The samples were pipetted into a PCR tube cap and then placed with inversion on a coverslip. The coverslips were mounted on the microscope and images were taken, keeping the pinhole equal to 1 AU. The laser power and the gain were adjusted in a way that best images could be obtained.

DLS (Dynamic Light Scattering)

Particle size analysis of aqueous solutions of polymerosomes was performed by dynamic light scattering using a Malvern Zetasizer Nano. Data analysis is based on non negative least squares (NNLS) method.

HPLC (High Pressure Liquid Chromatography)

The reverse phase columns were kept at a temperature of 50ºC throughout the purification. The aqueous phase used was TEAA 100mM and the organic phase was acetonitrile. A gradient of the organic phase was used from 10% to 100% acetonitrile in a time interval of 20 minutes. In the case of the palmitate-arachidic acid modification, this wasn’t sufficient for the product to elute so a gradient with methanol.

UPLC (Ultrahigh Pressure Liquid Chromatography)

The detection of reaction products were performed with ACQUITY Ultra Performance Liquid Chromatography system, using BEH C18 columns. The detection limit of this system is 10 uM. The retention time for the substrate and the reaction products were obtained, after elution with water.For the cases when UPLC measuremnts were performed with nanoreactors, filtration of the samples were performed with 0,22 um filters in order to remove the polymersomes from the solution.

Mass spectrometry

Samples were analyzed with an electrospray mass spectrometry device with a quadrupole detection system coupled to a UPLC (ultrahigh pressure liquid chromatography) purification system.

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