Experiment 4 - Synthesis of Barrel and Biosensor
AimTo structually characterise our cooperative biosensor.
BackgroundHaving designed and synthesised the sequences required for our barrel and cooperative biosensor, and having optimised a method for generating sufficient ssDNA scaffold, we were ready to build the thing!
Our structural characterisation of these DNA origami structures involves two kinds of evidence - agarose gels and small-angle X-ray scattering:
- Gels are obviously limited in the kinds of information they can provide, however, the fact that linear and circularised DNA travel at different rates in agarose gels meant that it was possible to test our assembly of the barrel by carefully omitting those staples that hold the linear ssDNA scaffold in a cylindrical shape, thus leading to an observable difference between 'barrels' and 'broken barrels'.
- We were lucky enough to be sponsored by the Australian Synchrotron in the form of beamtime. This experiment involved first generating a realistic atom-based model of our cooperative biosensor, then forward-predicting a range X-ray scattering results, including all kinds of possible contortions and unintended imperfections in our barrel. This helped us understand and manipulate the SAXS data.
Conducting these experiments involved three different types of protocol, each covered separately below:
- Small-angle X-ray scattering
Methods and MaterialsAfter generating sufficiently high-yields of our custom DNA scaffold , we confirmed the purity on an agarose gel and estimated the concentration by Nanodrop.
At the same time, we combined all our synthesised oligos into a common mix wherever possible. This meant we used:
- a staple mix (12 oligos) to assemble the barrel at the core of our biosensor,
- a sensor mix (36 oligos) to assemble the switches on the outside of our barrel,
- three band mixes (24 oligos each) to assemble the different cooperative biosensors with different functionality.
These mixes were created by adding 2ul of each oligo at 500µM into their common mix. This was mixed thoroughly by pipetting and centrifuged at ~20,000g for 30 minutes to pellet any aggregate. The supernatant was removed and the concentration estimated by Nanodrop.
We then calculated the volumes of the scaffold and various staple mixes required to synthesise the biosensor at a particular concentration depending on the experiment required. For example Small Angle X-ray Scattering requires a high concentration of about 500ng/uL of sensor after 5 rounds of purification through a sephacryl S-300 High Resolution gel matrix. We combined the scaffold and staples in a single PCR tube and synthesised them on a thermocycler by heating to 90˚C and cooling back to room temperatature by 0.5˚C/min.
We initially assembled the barrel at the core of our cooperative molecular biosensor. To get some indication that it had indeed circularised to form a barrel, we included a synthesis of a 'broken' barrel that was missing a set of staples joined two ends of the barrel.
ResultsWe demonstrated that our core barrel could be assembled using our custom scaffold and that an annealing step was necessary to achieve self-assembly of our structure in a reasonable time frame. Additionally, the full and partial synthesis constructs consistently ran to a different length in agarose gels, indicating that they had different structures.
Fig 1. The final two wells of this gel show the assembled barrel at the core of our cooperative biosensor. 'Neat' indicates what happens when the staples are added to the scaffold and left at room temperature, whereas 'anneal' shows what happens after heating and cooling the mixture. Note that the two types of barrel run to a slightly different length, which indicates what happens when we change a 'barrel' into a 'tile' by removing those staples that connect the opposite ends of our scaffold strand.
Purification of Cooperative biosensor
AimTo purify the assembled biosensor from the excess staples used to anneal and store it.
BackgroundIt is believed that yields of DNA origami structures are maximised when a large, single scaffold strand is the limiting component, and an excess of staple strands are used to fold it. The use of a single limiting strand that traverses the entirety of the desired structure and a vast excess of smaller strands reduces the possibility of partially-assembled structures using up all the material. This synthesis strategy results in fully assembled structures an a large amount of waste material. However, for further experiments, the excess staples must be removed as they would add extra noise in structural analyses and the presence of excess ’sensor strands' would confound our functional analyses of the cooperative biosensor.
Methods and MaterialsWe first tried purifying our constructs using a sephacryl S-300 High Resolution gel matrix. We loaded the S300 bead slurry in to 3x empty micro-centrifuge spin columns. The beads were equilibrated with 5 column volumes of synthesis buffer (1000g 1 min) and spun dry 1000g 4 min). 50µL of un-purified assembly was loaded onto the dry columns and spun through (1000g 1 min). This flow through loaded onto another column and repeated three times.
ResultsThis purification protocol has previously been successful for other DNA nano-structures with 32nt staples. However, presumably since the size exclusion limit of this gel was ~118bp while the largest of our staples was 86nt and in great excess, we were not able to separate these long staples. :(
Fig 2. We showed that despite the size exclusion limit of S-300 beads being twice the mass of our largest staples, we still couldn't completely purify our staples using sephracryl S-300.
Small Angle X-ray Scattering
AimsTo use Small Angle X-ray Scattering (SAXS) to experimentally acquire basic structural information about our cooperative molecular biosensor.
BackgroundWe were very fortunate to receive sponsorship from the Australian Synchrotron, providing us with the ability to carry out SAXS experiments to structural characterise our system. SAXS is a technique which utilises the small wavelength of x-rays to provide information on the nanometer scale about the structure of materials. When x-rays interact with electrons in the material of the sample, their path is bent or "scattered". By looking at this diffraction at small angles (of less than 10 degrees) it is possible to calculate the relative distribution of the atoms in the material. SAXS has a key advantages over other structural techniques such as x-ray crystallography as it does not require the material being investigated to be crystalline, and AFM and EM as the structure is in solution rather than fixed on a surface.
Methods and MaterialsIn order to prepare a sample for SAXS, a concentration of ideally 500 ng/uL is required with higher concentrations yielding stronger, more easily analysed signals. We synthesised and purified barrel and cooperative sensor samples as described above.
To analyse the pattern of X-ray scattering we produced a 3D model defining locations of hydrogen-bonded base-pair in each helix as we expected them scanned a range of spacings between helices. These models are used as an input to Crysol, a program that back-calculates scatter profiles from the scan of possible 3D structures. The experimental scatter plot is then matched to the best Crysol output, giving an indication that the corresponding model is representative of the actual structure. For instance, the image below describes the maximum and minimum possible dimensions for a barrel made out of our ssDNA scaffold.
Fig 3. Extremes of 3D model scan for the barrel.
Fig 4. 3D model of expected cooperative switch structure.
We successfully generated a set of predicted curves for our atomic models. One key outcome of this modelling process was the realisation that hollow structures produce the wave-like patterns described below -
Fig 5. Modelled SAXS Data for Barrel
We were successfully able to collect SAXS images for the barrel, however unfortunately due to a mistake made during the purification method, only a small amount of complete switch was able to be analysed leaving a complex scattering pattern which at present we have yet to resolve. The raw barrel data initially did not seem promising, as the intensity vs angle curve was more characteristic of a rod like structure. This led us to believe it was possible that this sample of barrel had become contaminated with impurities from the PCR product. This was of course, clearly observable in the gels of our ssDNA scaffold used for annealing the barrel. As a result we imaged the raw PCR product itself, and on subtraction from the barrel data were able to resolve a characteristic curve of a hollow structure, like our barrel.
Fig 6. Processed SAXS Data for Barrel
ConclusionWe are confident we were able to complete the construction of a cooperative biosensor, the combination of gel experiments that indicate specific assembly with SAXS data the clearly shows the formation of a hollow cavity containing structure indicate that our cooperative biosensor has successfully assembled into our design. However as of time of writing, due to time constraints, we have been unable to effectively characterise the tuning of the biosensor.
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