Methodology

METHODS

METHODS

PROTOCOLS

DNA ORIGAMI PREPARATION AND PURIFICATION

Synthesis:

  1. A portion of each staple was mixed in PCR tubes to make a single solution of the staple strands with the desired stock concentration (typically a final concentration of 0.2μM of each staple).

  2. A 10X stock solution of the origami buffer was prepared. 200mM Tris HCl (pH 7.6) containing 100mM MgCl2 and 10mM EDTA was used.

  3. The staples (5μL of 0.2μM stock), M13 (2.5μL of 0.1-μM stock), and origami buffer (2.5μL of 10× stock) were mixed. The final volume was adjusted to 25μL using Milli-Q water.

  4. The solution was shaken gently or vortex to ensure the homogeneity of the solution.

  5. The solution was annealed from 85° to 15°C at a rate of −1°C/min using a thermal cycler (in the original report, it was 95° to 20°C in <2 hr).

Purification:

  1. The excess staples were removed from the origami solution using gel filtration. The Sephacryl S-300 gel used contains 20% ethanol as a preservative which has to be removed. To facilitate this, the gel was re-suspended in ∼40 mL of the desired buffer (usually in 1× origami buffer; i.e., 20mM Tris HCl containing 10mM MgCl2 and 1mM EDTA) and kept in a shaking incubator overnight (∼128 rpm).

  2. The chromatographic column was packed with Sephacryl S-300 as follows:

    1. A small hole was poked in the bottom of a chromatographic column and placed in a clean 1.5mL micro-centrifuge tube.

    2. About 500μL Sephacryl S-300 solution was added and centrifuged for 3 min at 1000x g 15°C. The buffer solution was discarded and the steps were repeated (i.e., adding of the Sephacryl solution, centrifugation, and discarding of the buffer collected in the micro-centrifuge tube) until the column was packed to a little below its neck (∼1.5 cm in height).

    3. Centrifugation was done again but without the addition of Sephacryl to ensure that the column is sufficiently free from excess buffer.

  3. The micro-centrifuge tube was exchanged with a new one. Without disturbing the gel bed, the origami sample was directly applied onto the top centre of the gel bed carefully. The column was centrifuged for 3 min at 1000x g, 15°C.

  4. The staples-free, purified origami sample was collected in a micro-centrifuge tube.

Characterization of the dna origami:

TEM ANALYSIS:

  1. A sample was diluted to 10nM structure concentration.

  2. The diluted sample was incubated on a TEM grid for 1 minute.

  3. The grid was stained using uranyl formate for 40 seconds.

  4. The grids were imaged at 100 kV.

SEM ANALYSIS:

  1. The sample to be analysed was dried thoroughly.

  2. The dried sample was then mounted onto a SEM sample stub with a double sided sticky tape.

  3. The sample was then sputtered with gold (because magnetite is non-conductive) for around 65 seconds.

  4. The sample was then imaged using SEM instrument of magnification 10 lacs, voltage of 5-30kV, resolution 3nm and working distance of 3-30mm.

GEL ANALYSIS:

  1. The gel was prepared using 2% Agarose, buffer containing 44.5mM Tris Base, 44.5mM Boric acid, 11mM MgCl2, 1mM EDTA and 1µM Ethidium Bromide.

  2. The sample was loaded onto the gel and run at 90V for 90 minutes.

  3. The gel was then imaged using a GE Typhoon FLA 9500 scanner at 50 µm scanning resolution.

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SYNTHESIS OF SPIONS

Synthesis:

  1. 19.46 g (0.119 mol) of anhydrous FeCl3 was completely dissolved in 150 ml distilled water to prepare aqueous solution A.

  2. Further, 6.584 g (0.0396 mol) of Potassium Iodide was dissolved in 50.0 ml of distilled water to prepare aqueous solution B.

  3. Solutions A and B were then mixed together at room temperature, stirred and allowed to reach equilibrium for one hour.

  4. The precipitate of iodine was filtered out, washed with distilled water. The washing was added to the filtrate. The whole volume of filtrate was then hydrolysed using NaOH solution which was added drop - wise with continuous stirring until complete precipitation of the black magnetite was achieved (pH 9–11).

  5. The set-up was then left to settle down, filtered, washed with distilled water and dried at 250 °C.

  6. The dry yield was finally weighed.

Characterization of Spions:

SEM ANALYSIS:

  1. The sample to be analysed was dried thoroughly.

  2. The dried sample was then mounted onto a SEM sample stub with a double sided sticky tape.

  3. The sample was then sputtered with gold (because magnetite is non-conductive) for around 65 seconds.

  4. The sample was then imaged using SEM instrument of magnification 10 lacs, voltage of 5-30kV, resolution 3nm and working distance of 3-30mm.

FTIR ANALYSIS:

  1. Potassium Bromide and the sample to be analysed were taken in 10:1 ratio and mixed thoroughly using mortar and pestle.

  2. The sample was gently placed in a KBr Press instrument and a pressure of 6 tons was applied.

  3. The set up was allowed to stand for 2 minutes for the pellet to form.

  4. The pressure was released and the sample was carefully transferred to FTIR sample holder.

  5. FTIR was performed and the graph was obtained.

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SYNTHESIS OF PEI (Polyethyleneimine) COATED SPIONS

Synthesis:

  1. 10 % PEI in water (w/v), pH 7.9 was prepared by weighing 10 g of PEI (molecular weight of 25 kDa branched, Sigma Aldrich) and dissolving in 75 ml of H2O. pH was adjusted to 7.9 with concentrated HCl and volume was made upto 100ml using water. The PEI solution was filtered through a 0.22 μm nitrocellulose filter and stored at 4 °C.

  2. 100 µL of PEI solution and 10 mg SPIONs were mechanically mixed in 60 mL water for 30 min.

  3. 1 mL of H2O2 was added, and then the mixture was irradiated by two 6W lamps for 30 min and N2 was bubbled throughout the preparation process at 2kg/cm2 pressure.

  4. The PEI-SPIONs were collected with a permanent magnet and washed with deionized water for several times and at last re-dispersed in deionized water.

Characterization of SPIONS coated with PEI:

SEM ANALYSIS:

  1. The sample to be analysed was dried thoroughly.

  2. The dried sample was then mounted onto a SEM sample stub with a double sided sticky tape.

  3. The sample was then sputtered with gold (because magnetite is non-conductive) for around 65 seconds.

  4. The sample was then imaged using SEM instrument of magnification 10 lacs, voltage of 5-30kV, resolution 3nm and working distance of 3-30mm.

FTIR ANALYSIS:

  1. Potassium Bromide and the sample to be analysed were taken in 10:1 ratio and mixed thoroughly using mortar and pestle.

  2. The sample was gently placed in a KBr Press instrument and a pressure of 6 tons was applied.

  3. The set up was allowed to stand for 2 minutes for the pellet to form.

  4. The pressure was released and the sample was carefully transferred to FTIR sample holder.

  5. FTIR was performed and the graph was obtained.

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ATTACHMENT OF PEI-SPIONS TO DNA

Synthesis:

  1. DNA at a concentration of 10 μg/ml in PBS (pH 7.4) was mixed with SPION/PEI complexes at N/P ratio of 10 (i.e., the molar ratio of PEI nitrogen to DNA phosphate).

  2. For the N/P ratio, appropriate amount of SPIONs/ PEI was mixed with 0.5 μg DNA in 25 μl PBS.

  3. SPION/PEI/DNA polyplex solutions were incubated at 37 °C for 30 min.

GEL SHIFT:

  1. 0.8 g of agarose was measured and poured into a flask along with 100ml of 1X TAE buffer.

  2. Agarose was boiled with swirling on a heater till a clear solution was obtained.

  3. The solution was allowed to cool to a temperature of about 50-55ºC.

  4. EtBr to a final concentration of approximately 0.5 μg/ml was added.

  5. The ends of the casting tray were sealed with two layers of tape and the combs were placed in the gel-casting tray.

  6. The melted agarose solution was poured into the casting tray and cooled until it solidified.

  7. The combs were then pulled out, tape was removed and the gel was placed in the electrophoresis chamber. TAE buffer was added such that there is about 2–3 mm of buffer over the gel.

  8. 5–6 μl of 6X sample loading buffer was added to each of 25 μl SPION/ PEI/DNA polyplexes.

  9. 20 μl of each sample/loading buffer mixture was pipetted into separate wells in the gel.

  10. Electrophoresis was run at 100 V for 90 min and then visualized using a UV illuminator.

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MRI PROTOCOL

Culture of cells:

  1. Fertilized chicken eggs were collected and the embryo was isolated, it was placed in saline solution to sustain the cells.

  2. Egg shell membrane was also isolated and placed in a clean petri plate.

  3. The tissues were isolated till they form a layer around 1cm in height.

MRI:

  1. The SPION-DNA complex was injected in the culture of cells.

  2. The petri plate containing the cells were placed in the MRI chamber.

  3. The MRI used is a Philips Ingenia 1.5T MR system.

  4. Survey images of the sample was taken to better understand the terrain of cells.

  5. Different MRI procedures like T1 weighted imaging, T2 weighted imagine, Proton Diffusion Weighted Imaging and BLISS Imaging were conducted.

  6. Dynamic imaging was done to improve the quality of the image.

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EQUIPMENTS AND SOFTWARE

SCANNING ELECTRON MICROSCOPE:

SCANNING ELECTRON MICROSCOPE:

The Scanning Electron Microscopy (SEM) is used to produce images by scanning it with a beam of electrons. SEM is used to visualize the synthesized SPIONS. SEM can achieve resolution better than 1 nanometer and are imaged under vacuum.

ULTRASONICATOR:

Sonication refers to application of sound energy to agitate particles in a sample, for various purposes. Ultrasonicator probe is very effective in processing nanoparticles. It is standard equipment for dispersion, de-agglomeration, size reduction of particles and surface functionalization. Ultrasonic frequencies (>20 kHz) are usually used.

ULTRASONICATOR

FTIR:

FTIR

Fourier Transform Infrared Spectroscopy(FTIR) is a technique that provides an infrared spectrum of absorption or emission of a solid, liquid or gas. The FTIR generates spectra with patterns that provide structural insights. Since FTIR gives quantitative information such as additives and/or contaminants, we have used this to characterize SPIONS.

GEL ELECTROPHORESIS:

Electrophoresis is a technique employed to separate molecules based on their size. Smaller molecules migrate faster and the larger ones take time and thus cover lesser distance. This principle is adopted to test the attachment of SPIONS-PEI to DNA.

GEL ELECTROPHORESIS

HOT AIR OVEN:

HOT AIR OVEN

Hot air ovens are used to sterilize using dry heat. It works on the principle off forced circulation of hot air inside an oven chamber. 250 degrees Celsius is the most preferred range. The drying of SPIONS requires high temperature and is attained using this equipment.

NITROGEN PURGING APPARATUS:

NITROGEN PURGING APPARATUS

Nitrogen purging is an industry standard technique for the replacement of a hazardous or undesirable atmosphere with an inert dry atmosphere. Nitrogen is inert, non- reactive, and will displace the oxygen preventing any oxidation reaction. Polymer coating requires an inert atmosphere and is facilitated by nitrogen purging.

KBr PRESS:

KBr mini-hydraulic pellet press is used to prepare 1-7mm thick pellets for FTIR samples. A pressure of 6-8 tons are usually applied to the sample mixed with KBr for pelletization.

KBr PRESS

MRI SCANNER – 1.5T:

MRI SCANNER – 1.5T

An MRI scanner works by emitting a strong magnetic field that aligns the nucleic spin orientation of hydrogen atoms at a low energy state in a patient. An ideal standard for MRI in a clinical setting is a 1.5T MRI. It is used to guide and track the SPIONS in the body.

MICROCENTRIFUGE:

A microcentrifuge is an instrument that spins the liquid samples at very high speed. It works on the principle of sedimentation. It is used to separate liquids from solids. Microcentrifugation is used for smaller volumes of upto 2ml.

MICROCENTRIFUGE

GEL DOC:

MRI SCANNER – 1.5T

Gel doc is a gel imaging system used for imaging and documenting nucleic acids or protein suspended in agarose or acrylamide gels. It provides accurate and high quality images for analysis and interpretation of results. The instrument has been used to image the gel shift assay performed.

SOFTWARE:

  1. Cadnano Version 2.2.0.:
             Cadnano is a plugin that is used to design and create 3D DNA origami nanostructures. This user friendly software allows the creation of arbitrary designs and can display both staple strands and scaffolds. Integrated within Autodesk Maya, this plugin can also analyse the stability of the elements.


  2. UNITY Version 5.4.0. :
            Unity is one of the best cross-platform game engine used to develop video games, simulations and websites. It has the ability to target games on various platforms. The latest version has improved features like de-noising filter, editor etc. This software has been used to simulate our whole project.


  3. PROTEUS version 7.1. :
         Proteus is a circuit simulator. It efficiently simulates the interaction between microcontroller and any analog or digital electronics.Our nanocircuit is designed using this software.


MATERIALS

  • Anhydrous FeCl3

  • Potassium iodide

  • PEI- Polyethyleneimine

  • Agarose

  • Staple strands based on the origami design

  • M13mp18 single-stranded DNA (New England Biolabs, cat. no, N4040S)

  • Sephacryl S-300 (High resolution; GE Healthcare, cat. no., 17-0599-01)

  • PCR tubes

  • Micro Bio-Spin chromatography columns (Bio-Rad, cat. no. 732-6204)

  • 1.5mL microcentrifuge tubes

  • Mica plate

  • 25% Ammonia solution

  • Hydrogen Peroxide- H2O2

  • DNA

  • Phosphate Buffer Saline- pH 7.4

  • 1X TAE buffer

  • Ethidium Bromide

  • Loading buffer

  • Sephacryl S-300 solution

  • 10× origami buffer (200mM Tris HCl, 100mM MgCl2, 10mM EDTA)