Team:EPFL/Results/Aptamers2
What was accomplished ?
Magnetic beads have allowed us to:
- Confirm that thrombin and PDGF can successfully be bound by 2 different aptamers simultaneously
- Prove that fluorescence detection is a viable testing method
- Prove that thrombin detection can be decreased to at least a 50 nM concentration
- Prove that the entire aptamer 1-target protein-aptamer 2 with trigger complex can successfully trigger toeholds in lysate
- Prove that proteins in their naturally occurring form can be isolated in cell free medium and subsequently detected
Thrombin aptamer 1 (Cy5) and Thrombin aptamer 2 (Cy3) double binding assay with biotinylated Human a-Thrombin
The data displayed in Figures 1a and 1b, obtained from a magnetic bead pulldown assay[1] shows that both aptamers bind to thrombin. The beads were coated by re-suspension in a 1 µM solution of biotinylated thrombin. The fluorescence signal was measured periodically for 90 minutes and the values shown are an average of the recorded signals. Due to the time necessary to perform the washing steps in the pull-down assay, the beads in the sandwich assay spent less time in contact with Thrombin aptamer 1, which could explain the lower fluorescence signal. Moreover, due to the regularity of the fluorescence levels over time, the necessity for long analysis periods is negated, which effectively reduces the time necessary for a complete assay.
The Cy3 fluorescence levels were measured using a plate-reader with excitation and emission spectrums set at 549 nm and 571 nm respectively. In the double bind assay (labeled Cy3 + Cy5 apt), both aptamers are bound to the thrombin and emit fluorescence. The turquoise bar labeled No Thr is the no thrombin control, meaning that the fluorescence displayed is due to unspecific binding. Finally, the Cy3 apt and Cy5 apt controls respectively represent assays where the Cy3 and Cy5 labeled aptamers were added to the beads.
Cy5 fluorescence levels were measured at wavelengths of 647 nm and 668 nm for excitation and emission respectively. The legend in this graph is the same as in Figure 1a.
Titration of Human α-Thrombin
In this experiment, several dilutions of Human a-Thrombin were made. Using magnetic beads to perform a single bind pulldown assay, we detected the lowest concentration of thrombin that can be bound by a Cy3 labeled aptamer 2. As can be seen in the figure below (Fig. 2), the lowest limit of detection of thrombin is 50nM in our system. We are likely limited by the fluorescence detection method used and the possibility that there is non-specific binding of the beads by labeled aptamers. Thus there is a potential to optimize the assay in order to detect lower concentrations of target proteins and improve upon the detection limit achieved with the microfluidic assays.
The Cy3 fluorescence levels were measured using a plate-reader with excitation and emission spectrums set at 549 nm and 571 nm respectively. The legend indicates the concentration of thrombin that was added to the beads and the control lacks thrombin altogether.
Triggering of the toehold using a magnetic bead sandwich assay
We performed magnetic bead pulldown assays in order to determine whether or not such an assay had the capacity to trigger the Zika toehold 27B in sufficient amounts to see a difference. After a pulldown assay where biotinylated PDGF aptamer 1, PDGF and PDGF aptamer 2 extended with a trigger sequence were added consecutively, we resuspended the beads in lysate containing the DNA encoding the Zika toehold 27B uphold of the LacZ gene. After 5 hours of incubation, the beads were then separated from the lysate, which was deposited into a micro-well plate for subsequent absorbance measurements.
From the data extracted, we can see that the absorbance at 595 nm (optimal absorbance to measure the substrate turnover of β-Galactosidase) increases more or less correctly with the different types of assays performed. As we can see the sandwich assay does indeed express a higher absorbance of β-Galactosidase than controls without PDGF and with or without trigger. Yet, in the first experiment, we can see a problem as the control without PDGF and trigger expresses a higher absorbance than that with trigger (Fig. 3a). However, this discrepancy disappeared when we repeated the assay (Fig. 3b) with the toehold upstream of the LacZ gene rather than using the toehold upstream of the LacZ-α gene, which was used in the first experiment. Hence, we can say, with the available data, that magnetic bead sandwich assays inducing toehold triggering in lysate are a viable option to detect naturally occurring proteins, proving that our initial hypothesis was correct, even though further testing is necessary to further characterize this process.
The absorbance of β-Gal was measured using a plate-reader at a 595 nm wavelength (maximum absorbance of β-Gal). This histogram shows the difference in absorbance between the positive and negative controls (+ control, - control, resp.), the sandwich assay and its respective controls. The positive and negative controls contain lysate with toehold, with or without trigger respectively, but said trigger was directly added to the solution and not the result of a pulldown assay. On the other hand, the sandwich assay controls are labeled in accordance to the addition or not of trigger and PDGF. Hence, the sandwich assay control without PDGF and trigger is labeled No trigger, no PDGF, whereas that without PDGF but where trigger was added is labeled Trigger, no PDGF. Finally, the sandwich assay containing all components for a successful detection of PDGF is labeled PDGF + trigger.
The graph shows the difference in absorbance between the complete sandwich assay (PDGF + trigger), the control where PDGF was not added (Trigger, no PDGF) and the control where neither aptamer 2 extended with trigger nor PDGF were added (No trigger, no PDGF).
