DNA nanopores self-assemble from synthetic single stranded sequences of DNA
(oligonucleotides). Oligonucleotides are designed using computer software
(cadnano) to fold into specific 3D structures.
We designed DNA nanopores as hexameric hollow barrels composed of six double
stranded DNA helices. We chose to build this nanopore as this archetypical
design has been independently described twice
(Langecker et al., 2012; Burns et al., 2013)
and has been recently demonstrated to embed in the membranes of liposomes
(Burns et al., 2016). Four of the six helices are equipped with cholesterol
anchors, in order to puncture lipid bilayers and allow the nanopores to
embed across the hydrophobic membranes.
List of ingredients:
1nM of oligonucleotides (ordered from Integrated DNA Technologies, IDT, as in the table below):
The DNA nanopores are assembled from oligonucleotides ordered from Integrated DNA Technologies. Resuspend lyophilised DNA in nuclease free water to a stock concentration of 100μM.
Prepare an equimolar mixture of DNA oligonucleotides (1nmol each, dissolved in buffer A 0.3M KCl, 15mM Tris pH 8.0; total volume 1,000μL) at room temperature.
Incubate at 95°C for 2 min and gently cool to 20°C at a rate of 5°C per min in a PCR thermocycler.
List of ingredients:
1nM of oligonucleotides (ordered from Integrated DNA Technologies, IDT, as in the table below:
The DNA nanopores are assembled from oligonucleotides ordered from Integrated DNA Technologies. Resuspend lyophilised DNA in nuclease free water to a stock concentration of 100uM.
Prepare an equimolar mixture of DNA oligonucleotides (1nmol each, dissolved in buffer A: 0.3M KCl, 15mM Tris pH 8.0; total volume 1,000μL) at room temperature.
Incubate at 95°C for 2 min and gently cool to 20°C at a rate of 5°C per min in a PCR thermocycler.
Gel electrophoresis assays were used to test whether (1) the nanopores had been assembled,
(2) strand displacement reactions were functional, and (3) the nanopores were being opened by the key.
By visualising bands which correspond to particular DNA complexes, gel electrophoresis
provides insight into whether the modelled DNA-DNA and DNA-protein interactions were taking place.
List of ingredients:
DNA samples
Agarose
1X TBE or 10X TBE as preferred
Deionised water
Conical flask
SybrGold
Microwave oven
Mould for the gel
Procedure:
Dissolve agar into 1X TBE in a conical flask
Heat the conical flask in a microwave for a few minutes until the fluid is entirely clear. Be careful to not superheat it as this can cause serious injury. The flask is very hot at this stage, so using heat resistant gloves to hold it is recommended.
5µl of SybrGold is added to every 50ml of agar-TBE buffer in the heated conical flask. This is poured into the mould with the comb. The mould is left immersed in 150ml 1X TBE until it solidifies. This usually takes about an hour.
Gel electrophoresis assays were used to test whether (1) the nanopores had been assembled,
(2) strand displacement reactions were functional, and (3) that the nanopore was being opened.
By visualising bands which correspond to particular DNA complexes, gel electrophoresis is capable of
providing insight into whether the modelled DNA-DNA and DNA-protein interactions were taking place.
List of ingredients:
Prepared agarose gel
Running gel apparatus (horizontal tank, lid)
Power source and cables
ThermoFisher SybrGold (or equivalent)
Gel loading dye
UV/blue light illuminator
Typhoon FLA 9500 gel imager
Samples to be run
1X TBE running buffer
NEB low and/or high molecular weight ladder(s)
Milli-Q (i.e. ultrapure) water
Procedure:
Gently remove the gel from the mould and transfer into the BioRad tank. Gently remove the comb.
Fill the tank with 1X TBE buffer so that it completely submerges the gel.
Stock DNA at 1µM was diluted using Milli-Q water: 10µL DNA sample + 2µL water + 3µL gel loading dye was pipetted into an eppendorf. 10µL of the resultant solution was loaded into the wells. Make sure to include at least 1 ladder: 0.5µL of the ladder + 3µL of gel loading dye + 11.5µL of water was pipetted into an eppendorf. 10µL of the resultant solution was added to the wells. The stock gel loading dye was at x5 concentration.
Load the wells with 10µl of the prepared sample by hovering the pipette tip just above the desired well. Ensure that you include a ladder (also mixed with gel loading dye).
Close the tank with its lid and run the gel at 70V for 1 hour. When the bands have travelled a sufficient distance, the power source can be switched off.
Gently lift the gel out of the tank and visualise under UV or blue light.
Building Liposomes
Liposomes (vesicles) are membrane-enclosed spheres that transport molecular cargo within and between cells. We used large unilamellar vesicles (LUVs) with a diameter of 200nm as drug carriers.
The first step of LUV preparation consists of drying phospholipids into glass vials to form “lipid films”. These can be stored at 4°C and taken out for use as needed.
List of ingredients:
25 mg mL⁻¹ DOPC (Avanti Polar Lipids)
25 mg mL⁻¹ DOPE (Avanti Polar Lipids)
Chloroform
Nitrogen gas
Procedure:
Add DOPC and DOPE into a vial in a 3:7 DOPE:DOPC ratio and make the solution up to the desired volume with chloroform. In this experiment, 30µL of DOPE, 70µL of DOPC and an additional 100mL of chloroform were used
The vial is dried using nitrogen gas and left in a vacuum oven overnight without any heat, at 400 bars of pressure.
Liposomes are formed by hydrating the dried phospholipids in an organic solvent. During this step, it is possible to fill the
liposomes with hydrophilic molecular cargoes (such as fluorophores and drugs) and embed DNA nanopores onto the membranes. A fresh
batch of hydrated liposomes was typically prepared on the same day of experimentation, whereas vials with lipid films were prepared
up to 3 weeks in advance and stored at room temperature.
List of ingredients:
Methanol (10µL)
DNA nanopores (150µl, 1µM in Buffer A)
Buffer A (0.3M KCl, 15mM TRIS, pH8)
Sulforhodamine B (200mM in Buffer A)
Procedure:
DOPE and DOPC dissolved in chloroform are added to a vial in a 3:7 ratio
50µL of 200mM SRB in buffer A, 150µL of a 1µM solution of DNA nanopores in buffer A, and 10µL of methanol are added to the vial. Two separate phases should be visible.
The apolar phase (chloroform) is evaporated in a vacuum rotary evaporator at 200rpm.
Extrusion is required to convert the multilamellar (i.e. consist of many lipid bilayers)
vesicles into unilamellar vesicles. This can improve the efficiency of incorporating fluorophores and nanopores.
List of ingredients:
Extruder (Avanti mini extruder kit)
Polycarbonate filter (0.2 µm)
Filter supports
Hydrated vesicles (from step before)
Procedure:
Assemble the extruder following Avanti instructions
With a glass syringe draw the hydrated LUVs solution from the glass vial
Extrude following the extruder-kit instructions. Make sure to exchange the LUVs solution between the syringes for an odd number of times, and a minimum of 11.
Collect the extruded solution in a suitably-labeled Eppendorf tube
The extruded solution now contains LUVs filled with fluorophores and DNA
nanopores embedded on the membrane. However, many fluorophore molecules remain unencapsulated. To purify LUVs from
unincorporated fluorophores, size-exclusion chromatography by gel filtration is performed.
List of ingredients:
Buffer A (0.3M KCl, 15mM TRIS, pH8)
Sephadex G-100
Sephadex Columns
Procedure:
0.8g of sephadex powder is dissolved in 24mL of buffer A in a 50mL Falcon tube.
It is left to settle for 3 hours at room temperature or for 1 hour at 65°C in a vacuum oven.
The tip of the column is cut with scissors. A filter is added to the bottom of the column and sephadex is poured on top of it. The number of air bubbles should be minimized. The column is kept open. A waste beaker is placed underneath.
As the sephadex settles, the height of the column should be monitored. Top up sephadex as needed to achieve the appropriate column height.
Once the sephadex solidifies to the point where the column height does not drop any further, even when elution buffer is added, another filter is placed on top of it.
Pre-equilibrate the column with elution buffer.
Pipette a solution of liposomes on top of the column and elute with elution buffer. 1mL aliquots are collected in Eppendorf™ test tubes.
Building Peptide-Oligonucleotide Conjugates (POCs)
After succesfully demonstrating that the DNA nanopores open upon detection of the key input and allow the passage of molecular cargoes across the membrane, we aimed to demonstrate that the nanopores can be used to sense protein substrates. For this we designed nanoscale Peptide-Oligonucleotide Conjugates (POCs), covalent constructs that link oligodeoxinucleotides to a synthetic peptide sequence. These materials merge the programmable self-assembly of oligonucleotides with the bioactivity and chemical diversity of polypeptides (MacCullogh et al., 2019).
POCs are composed of peptide sequences flanked by tandem DNA sequences that form defined tertiary structures.
The ultimate goal of POCs is to leverage the unique advantages of each biomolecule into a chimeric molecule that allows DNA nanopores to sense proteins.
POCs were assembled by separate synthesis and purification of each component (peptides and oligonucleotides) followed by their coupling in solution using the heterobifunctional amine-to-sulfhydryl cross-linker succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), following the protocol described by Williams and Chaput, 2011.
Oligonucleotides were designed to have extra amine (NH2) groups at their 3’ or 5' ends (ordered from Integrated DNA Technologies)
Peptide sequences (with 2 terminal cysteines at the N- and C- termini)
Description
Sequence
Peptide linker
Cys-Gly-Gly-Gly-Lys-Gly-Gly-Gly-Cys
100 mM KH2OPO4 buffer, pH 7.2
SMCC
Acetonitrile
Conjugate SMCC to DNA oligonucleotide:
Resuspend the purified oligonucleotide (key) in ultrapure water to reach a 100μM stock.
Transfer 20 nmoles of the oligonucleotide from step 1 to a new 1.5 mL tube.
Add 134μL of 100mM KH2PO4 buffer, pH 7.2 to the tube.
In a separate 1.5mL tube, dissolve 1mg of SMCC in 1mL of acetonitrile for a final concentration of 3mM.
Transfer 67.0μL of SMCC (200 nmoles) solution to the oligonucleotide solution prepared in step 4.
Vortex the solution vigorously for 5 seconds. Centrifuge the tube.
Allow the reaction to take place at room temperature for 30 minutes.
Repeat steps 1-7 for a second oligonucleotide (key blocker) to conjugate with SMCC.
Transfer the contents of the two oligonucleotide-SMCC containing tubes into a new 1.5mL tube.
Conjugate SMCC-oligonucleotide to cysteine-containing peptide:
Resuspend the peptide in water for a final concentration of 1 mg/mL solution.
Add 69.4μL (100 nmoles) of peptide to the tube in step 9 from the previous procedure.
Vortex the solution for 5 seconds.
Centrifuge the tube briefly.
Let the reaction take place at room temperature for at least 3 hours to obtain crude peptide-oligonucleotide conjugate (POCs) material.
Polyacrylamide gels are used to separate smaller fragments of DNA or reveal differences between lanes when the strands differ by a small number of base pairs. Polyacrylamide gels were made without any denaturing agent as described below.
List of ingredients:
1M Tris-HCl pH 8.8
Acrylamide/Bisacrylamide (30%/0.8% w/v)
10% (w/v) ammonium persulfate (AP)
TEMED
BioRad glass plates (one tall, one short per gel)
BioRad casting frame and casting stand
Isopropanol
Comb
Deionised water
Filter paper
Procedure:
Align the 2 glass plates together and insert them into the casting frame, secure the frame around the plates by clamping them. Suspend the apparatus from the casting stand.
Add some water in between the glass plates and test whether it leaks (it should not). If the test is successful, draw the water out with some filter paper.
Create a gel of the appropriate polyacrylamide strength using the table below as a guide. Note, add 10µL of TEMED last. TEMED enables the acrylamide to polymerise and if left to sit for a minute, could result in the gel solidifying too much to pipette within the falcon itself. Therefore, it is important to quickly pipette the gel into the glass mould after adding TEMED.
10% Gel
15% Gel
Acrylamide/Bis-acrylamide
3.4 ml
5 ml
Tris-HCl
6.49 ml
4.89 ml
AP
100 µl
100 µl
TEMED
10 µl
10 µl
Fill the glass plates with the gel upto a height of about 1cm beneath (what will later become) the lowest position of the comb.
Remove the bubbles and ensure a straight gel by freely adding isopropanol.
After waiting for about 30 minutes for the gel to solidify, pour off the isopropanol and then dry off any remnants of isopropanol using filter paper.
Now, the stacking gel can be cast. In a 50ml Falcon, combine 4.275ml 1M Tris-HCl, 0.67ml Acrylamide/Bis-acrylamide, 0.05ml AP. Lastly, add 5µL of TEMED. This will result in 5ml of stacking gel.
Add this up to the top of the glass plates and then insert the comb.
Allow the gel to settle for another 30 minutes before removing the comb.
List of ingredients:
Prepared polyacrylamide gel (PAGE)
Gel running apparatus (holder, cassette, vertical tank, lid)
A power source and cables
Glycerol
Native gel loading dye
1X or 10X TAE buffer
Prepared crude POC material
Low and/or high molecular weight ladder(s)
Procedure:
Remove the glass plates from the casting frame and load them into the BioRad running gel apparatus, in accordance with the manufacturer’s instructions. Briefly, keep the short glass facing inwards and slide the plates onto the BioRad holder. The holder can accommodate 2 sets of glass plates (i.e. 2 gels) at a time. Use a dummy mould (i.e. glass plates without any gel) if only running 1 gel. Place the loaded holder into the BioRad running cassette and clamp them into position.
Load this into the BioRad tank.
Fill the bottom reservoir of the gel apparatus with 1X TAE buffer. Remove any air bubbles trapped under the gel plates.
Pour 1X TAE buffer in the top reservoir of the electrophoresis apparatus to ~3 cm above the top of the gel.
Rinse the wells of the gel with 1X TAE buffer to remove any residual polyacrylamide.
Prepare the previously made crude POC material by adding 1/10 the volume of glycerol and 1/10 the volume of 10X TAE buffer to the assembled crude POC solution.
Add 3µL of native gel loading dye to 15µL of the POC solution
Load 18µL of the POC solution and of the low and high molecular weight ladders by inserting the tip of the pipette perpendicularly to the edge of the short glass plate, just above the desired well.
Connect the gel apparatus to the power supply and run the gel at 200V at 4 °C for 1 hour.
List of ingredients:
Prepared polyacrylamide gel (PAGE)
ThermoFisher SybrGold (or equivalent)
Milli-Q (i.e. ultrapure) water
Typhoon FLA 9000 gel imager
Procedure:
The gel is removed from the reactor and from the glass plate mould, carefully ensuring not to break the gel.
SybrGold binds the DNA, allowing it to be visualised. Dissolve 5µL of 1000X stock of SybrGold dissolved in 50ml of 1X TAE (or 10X TAE diluted in Milli-Q water). Immerse the gel in this for about 40 minutes on a shaker.
Place the gel on a UV illuminator for a quick analysis of the position of the DNA bands. For a better quality readout of the position of DNA bands, use an instrument such as the Typhoon FLA 9500TM gel imager. A “SYPRO Ruby” filter on this instrument gave an optimal readout.
