Biomod/2014/VCCRI/LabBook/Exp2

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EchiDNA 2014

Experiment 2 - Characterisation of Molecular Beacon

Aim

To build and characterise the re-designed molecular beacon, and specifically to:
  • optimise purity and yield of switch construction,
  • and investigate the sensitivity of molecular beacons by systematically varying clip strength and signal concentration.

Fig 1. Diagram of our redesigned modular molecular beacon, indicating the single letter abbreviations of subcomponents commonly used by Team EchiDNA.

Background

We re-designed conventional molecular beacons to suit them to a new application in our cooperative molecular biosensor. We needed to determine whether our stripped-down, modular version of molecular beacons really worked!

There were three key measures of whether our re-designed molecular beacons were a success:
  • Firstly, we had to see whether or not our de-constructed version of the single-stranded molecular beacon would still hybridise into a complex of four separate oligos with predictable efficiency.
  • Secondly, we had to determine whether the re-designed molecular beacon still functioned and identify the limits of the switch in terms of signal-to-noise and the dynamic range of biosensing activity.
  • Finally, we wanted to exploit the modularity of our design to fully explore and characterise the potential of the switch as a biosensing technology.
In the first case one of the simplest and most direct ways to observe assembly is simply to create samples where all the components of the molecular biosensor are isolated, and others where they are together. By applying an electric field over a non-denaturing poly-acrylamide gel (PAGE) containing the DNA samples, it was possible to directly observe the change in mass after mixing complementary oligos. It was also possible to observe the oligo-bound fluorophore changing in mass upon hybridisation. In the second and third cases, we moved directly towards fluorescence as a direct means of testing whether the redesigned molecular beacon worked as anticipated.

We actually iterated through this set of experiments twice, with two evolving versions of our molecular beacon. Through these experiments we found that our first design had two major problems, which we managed to overcome in the second design.
Fig 2. Overview of reaction with the first design of the single beacon.

Fig 3. Overview of reaction with the second design of the single beacon.

Methods and Materials


Confirming synthesis of molecular beacons by electrophoresis
It is possible to separate small molecules by size based on their movement through a gel. The molecules are forced to move by an electric field over the gel. We can use a non-denaturing acrylamide gel to separate nucleic acids, and it is one of the most direct ways to see whether we have annealed oligos into structures of different sizes.
  • Preparing the gel
    • Set-up gel mould as per instructions
    • Set the resolving gel
      • Add Tris-HCl, acrylamide and Milli-Q water
      • Once mixed, add APS and TEMED
      • Fill resolving between gel plates up to 1cm below wells
      • Seal with a small layer of 100% isopropanol (~200 ul) and allow to set (~30 mins)
    • Remove isopropanol by pouring out or using filter paper
    • Repeat the process of preparation for the stacking gel
      • Fill stacking gel almost to overflowing
      • Insert well combs at an angle to allow air to escape from the wells
      • Allow to set (~30 mins)
    • Gel can be stored in Milli-Q water or run immediately
  • Running the gel:
    • Set-up gel and electrode in get bath (if you are only running one gel, remember to add another electrode, completing the circuit)
    • Fill the space between electrodes and tank with TRIS-glycine buffer, and fill about half the bath (until electrodes are submerged)
    • Remove the combs carefully
    • Add sample
    • Run at 150V, 400W for 40 mins, until lower band of dye reaches ~1cm from the bottom of the gel
    • Gel can be visualised in two ways (or both sequentially)
      • Immediately visualise using FujiFilm FLA-5100 Imager at 473nm (Alexa Fluor 488 can be seen on fluorophore strands)
      • Incubate gel for ~10 minutes in SYBR Gold Bath (5ul SYBR Gold in 50ml TRIS-glycine). Mixing and under foil to protext SYBR Gold from light. Visualise using FujiFilm FLA-5100 Imager at 473nm


Confirming function by titrating signal against clip strength

We needed to observe the resulting fluorescence from varying the signal concentration, clip strength, and exposure time in order to fully characterise our re-designed molecular beacon. To do this we designed the following experiment which would exploit the modular clip length of our system.

Fig 5. Explanation of different clip strengths.

  • Preparation of Stock Solutions: All oligos are dissolved and diluted in synthesis buffer unless otherwise indicated. Prepare 10 uM working stock solutions of Flurophore, Quencher, and each Clip. Prepare working stock solutions of signal at 10 uM, 1 uM, 100 nM and 10 nM.
  • Preparation of Unclipped switch "BFQ": A total of 70 uL at 2.14 uM of unclipped switch was prepared by combining a 1.5 uL aliquot of 100 uM backbone with 16 uL of 10 uM flurophore, 16 uL of 10 uM quencher, and 36.5 uL of synthesis buffer. The mixture was left to stand at room tempreature for 15 minutes.
  • Preparation of Clipped Switches "BFQC": There are a total of 7 clips covering a range of lengths from 0-10 base pairs, as well as a "blank" no-clip experiment. The 70 uL solution of "BFQ" prepared above is seperated into eight lots of 8 uL aliquots, to which 18 uL of 10 uM of each clip (or buffer in the case of no clip) is added. A further 69 uL synthesis buffer is added to make the total volume up to 95 uL.
  • Addition of Signal: 10 uL of each complete sensor is aliquotted nine times onto a 96 well plate, creating a 9x8 grid. Signal is then added to each sample (see table below). The plate is then scanned at 5 minute intervals on the plate reader over a period of 12 hours.
Fig 6. Table of Signal additions for grid (note all volumes are in uL)




Results

Confirming Synthesis
From the polyacrylamide gel, stained with SYBR Gold, we are clearly able to identify the distinct bands of each individual components of the switch. Furthermore, we see a clear shifted band when any of the components of the molecular beacon are paired with the backbone (B) that threads through our entire re-designed molecular beacon. Finally, we are confident from this gel that we have assembled a switch that functions correctly - by comparing the final three wells we can infer that while both the clip and signal hybridise independently to the molecular beacon, the signal will outcompete the clip when both are present in the same assembly mix.
Fig 6. Polyacrylamide gel demonstrating successful assembly of switch with high efficiency.
Key: (F) Fluorophore, (S) Signal, (C) Clip, (B) Backbone), (Q) Quencher.





Confirming Function
Fig 2. Overview of reaction with the first design of the single beacon.

It was by conducting exactly this kind of exhaustive search through the parameter space of different clip strengths and signal concentrations that we identified the design faults in our first molecular beacon. Consider the graphic, which shows the effect of increasing signal concentration (left to right) on decreasing clip strength (front to rear). Note that the weakest clips have very high levels of fluorescence, higher in fact than the negative control, containing no clip. This led directly to the re-design of our molecular beacon.
Fig 7. Switch design #1. Fluorescence plate data over a range of clips and signals.

Fig 3. Overview of reaction with the second design of the single beacon.



After re-designing and heading back into the lab with the second design of our molecular beacon, we conducted the same analysis of clip strength of signal concentration. This time, however, we conducted a time series analysis. This allowed us to demonstrate that the stronger clips take longer to be displaced by the signal, but eventually end up demonstrating similar overall behaviour as the shorter clips. It was also this analysis which led us to demonstrate that the binding of the signal, thus changing the local environment around the fluorophore, has a greater effect on the intensity of fluorescence than does the quencher-fluorophore interaction. This can be concluded from the fact that at low concentrations of signal, the difference between clips is explained by whether or not the clip is long enough to hold the switch shut and bring the fluorophore and quencher into close proximity.
Fig 8. Switch design #2. A time-series analysis of fluorescence considering multiple different clip strengths over a range of signal concentration (varying from 1/64 to 64 x excess of signal:molecular beacon).

Conclusion

We have constructed a single modular non-cooperative DNA biosensor and functionally characterised its fluorescence. This process led to new and improved designs, while also teaching us more about fluorophores. This work brought us one step closer to building our cooperative molecular biosensor by demonstrating that we have the capacity to iteratively optimise our biosensor by exhaustively searching the parameter space of modular designs.


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