Experiment 1 - Characterisation of Fluorophores
AimTo familiarise ourselves with the detection of fluorophore-modified synthetic DNA.
BackgroundOur project used fluorophores to detect conformational changes in DNA. Before getting stuck into any fancy experiments with all our crazy designs, we thought it was worth checking that we could actually detect changes in fluorescence.
Fluorophores generally involve a specific arrangement of covalently bound atoms that can absorb light and re-emit it at a different wavelength. The changes in fluorescence we expected to observe were based on the known absorption and emission spectra for a set of well-characterised fluorophores and dyes - AlexaFluor 488 and Black Hole Quencher, purchased from Integrated DNA Technologies (IDT). Specifically, it was anticipated that decreasing the concentration of a fluorophore would linearly decrease the intensity of fluorescence, until such a low concentration is reached that fluorescence of these molecules could not be detected above the background fluorescence of a solvent like water.
Identifying the limits of detection of fluorescence was necessary in order to inform future experiments, and also to determine whether we could feasibly use fluorescence as the output for our biosensor.
Methods and Materials
Investigating the limits of detection of fluorescence
- In our first experiment we checked that we could detect the Alexa488 dye. We used this particular unmodified fluorophore as it was the one we anticipated using in later experiments.
- We created a dilution series of the fluorophore over a wide range of concentrations (0.25 pMol to 128 uMol). Our 5 mM stock solution of Alexa488 was diluted in Synthesis Buffer (33nM Tris acetate, 12.5 nM Mg(OAc)2) to a working stock of 5uM, from which 1 mL of 128 nM was produced and then serial 2x dilution in buffer were produced down to 1/1024 or approximately 0.25 pMol.
- 100uL of each sample was transferred into wells and the entire well plate was imaged using a Pherastar Plate Reader.
Understanding the effect of the local chemical environment on fluorescence.
Fig 1. Different chemical environments may affect the intensity of fluorescence.
- We wanted to define precisely how the presence of single-stranded or double-stranded DNA would affect the intensity of fluorescence. This was necessary before we could say confidently that the changes in fluorescence we observed in future experiments were caused by conformational changes in DNA, and not simply the presence or absence of DNA.
- We hybridised different oligos of DNA by mixing them as outlined to the right and leaving the solutions to hybridise for half an hour at room temperature.
ProtocolIn order to test these effects, the following reagents were added to microcentrifuge tubes:
B stands for 'Backbone' and S stands for 'Signal'. These different oligos in different combinations create the different conditions for this experiment. The strands were given time to anneal before observing using the Pherastar plate reader.
ResultsThrough a simple dilution experiment, carried out in triplicate, we were able to identify the lowest concentration of fluorophore that we could reliably detect using our available resources. This result shaped future experiments as it indicated the concentration of fluorophore we needed to use to get a reliable signal-to-noise ratio without wasting expensive fluorophore. Furthermore, it also indicated the ultimately limited sensitivity of fluorescent output of molecular beacons, which stimulated us to think of alternate outputs for our cooperative molecular biosensor.
Fig 3. Results of the standard curve characterising our fluorophores. Our agreed-upon minimum detectable concentration was 0.125nM.
Through another simple experiment we were able to show that fluorophore-modified oligos are more complex than we had anticipated. Changing the chemical environment around the fluorophore had a significant impact on the intensity of fluorescence. Who knows! It may be that this effect, rather than proximity to a quencher, is also part of the mechanism underpinning conventional molecular beacons.
Fig 4. Results for experiment investigating effects of local chemical environment on fluorophore signal intensity.
ConclusionsThis set of experiments helped us understand how fluorophores work, guided our future designs, and taught us how to use and calibrate the relevant tools for observation and analysis of fluorescence. This was the first in a series of steps leading us towards characterising molecular beacons and building a cooperative molecular biosensor.
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