Team:TU Eindhoven/Measurement
Measurement
Measurement
One of the main goals within our project is to develop a detection method to detect specific dsDNA sequences without using any complex laboratory equipment. This method should be modular to make sure any dsDNA target could be detected. Furthermore, specific detection should be possible in a short period of time. We succeeded in designing and developing a dsDNA detection method called paired dCas9-Split-NanoLuc system. The protein dCas9 is used to target unique DNA sequences. Guide-RNA (gRNA) enables dCas9 to target a specific sequence. Unique gRNA sequences can be determined with our dCas9 gRNA finder tool. Any dsDNA sequence can be detected by designing the matching gRNA, which makes this system modular.
The paired dCas9-Split-NanoLuc system is based on two dCas9 proteins that bind in close proximity of each other, which enables the attached split luciferases to form a complex and emit blue light (Figure 1). This paired dCas9-Split-NanoLuc system consists of two composite parts; BBa_K3168004 and BBa_K3168005.
Figure 1: Schematic representation of the dCas9-Split-NanoLuc system.
By measuring the bioluminescence intensity with varying target DNA concentration, the limit of detection for our dCas9-Split-NanoLuc system was determined (Figure 2). From these measurements it was concluded that our system is able to detect target DNA concentrations up to 10 pM with a sensor protein concentration of 2 nM and an interspace distance of 20 base pairs. It is even expected that the limit of detection for our system lies lower than the 10 pM presented because this limit was determined in sub-optimal conditions. I.e. the measurements were performed with an interspace distance of 20 base pairs while other measurements have shown that the optimal interspace distance lies at 70 basepairs (see Results). Furthermore, this limit of detection was determined with one dCas9-Split-NanoLuc recognizing one site on one target while with multiple dCas9-Split-NanoLuc recognizing more sites on one target resulted in a significant enhancement in bioluminescent signal.
Figure 2: Bioluminescence intensity with varying target DNA concentration at a constant 2 nM sensor protein concentration with interspace distance 20.
Calibrator luciferase NanoLuc-mNeonGreen
The paired dCas9-Split-Nanoluc system relies solely on measuring intensity changes. This hinders measurements of this system that are independent of conditions such as time, concentration, pH and temperature. To overcome this problem, the NanoLuc-mNeonGreen (BBa_K3168009) reporter protein has been designed. The function relies on Bioluminescence Resonance Energy Transfer (BRET), where NanoLuc acts as the donor and mNeonGreen as acceptor (Figure 3). NanoLuc and NanoLuc-mNeonGreen both convert the same substrate and have a similar stability over time and in various measurement conditions. Therefore, NanoLuc-mNeonGreen enables ratiometric measurements where NanoLuc-mNeonGreen acts as a calibrator luciferase, which allows time and concentration independent measurements. This results in measurements that are more reliable than non-ratiometric measurements due to the independence of these conditions. The concept can be implemented easily when performing measurements with NanoLuc and wishing to do ratiometric calibration measurements.
Figure 3: Schematic representation of the BRET between the donor NanoLuc and the acceptor mNeonGreen.
Time and concentration independent measurements
To prove that condition-independent ratiometric measurements could be performed with NanoLuc-mNeonGreen as calibrator luciferase, measurements with different concentrations of NanoLuc and NanoLuc-mNeonGreen were executed as well as measurements over time (Figure 4 & 5). The measurements with varying concentrations of NanoLuc and the calibrator NanoLuc-mNeonGreen indicate that the ratio (being ±4.5) between these two is constant for different concentrations, showing that concentration independent measurements can be performed with use of the calibrator luciferase. The measurements over time indicate that the ratio (being ±3.5) between NanoLuc and the calibrator NanoLuc-mNeonGreen stays constant over time, enabling time independent measurements.
Figure 4: Luminescence intensity at emission maximum for NanoLuc (460 nm) and NanoLuc-mNeonGreen (517 nm) for increasing concentrations of both proteins.
Figure 5: Luminescence intensity at emission maximum for NanoLuc (460 nm) and NanoLuc-mNeonGreen (517 nm) over time.
Ratiometric measurements of paired dCas9-Split-Nanoluc
Before executing measurements with NanoLuc-mNeonGreen as calibrator luciferase for measurements with the paired dCas9-Split-NanoLuc system, the ratio of light intensity between the two needs to be determined. For the calibration measurements, the intensity of the calibrator luciferase should be around 10% of the intensity of the luciferase to facilitate measurements of concentrations that are ten times as high as well as ten times as low. For our paired dCas9-Split-NanoLuc system, the concentration of NanoLuc-mNeonGreen needs to be around a thousand fold lower than the concentration of dCas9-Split-NanoLuc to achieve this 10% intensity as can be seen in Figure 6, where the intensity of 2 pM NanoLuc-mNeonGreen (at its maximum emission wavelength of 517 nm) corresponds with 10% of the intensity of 2 nM dCas9-Split-NanoLuc (at 460 nm).
Figure 6: Relative bioluminescence for dCas9-Split-NanoLuc and different concentrations of NanoLuc-mNeonGreen.
After determining which concentration ratio was required to achieve the 10% intensity, we could perform ratiometric measurements with NanoLuc-mNeonGreen as calibrator luciferase for our dCas9-Split-NanoLuc system (Figure 7). By using the calibrator luciferase, a more reliable calibration curve is obtained, one that is independent of the measurement conditions. However, this experiment was only performed once and therefore the calibration curve could be optimized and more measurement points could be included. By optimizing and repeating the measurements, the error bars can be decreased as well. Nevertheless, a trend in the signal can be observed, which is similar to the trend observed when determining the limit of detection for the paired dCas9-Split-NanoLuc system (Figure 2).
Figure 7: Target DNA calibration curve of the dCas9-Split-NanoLuc (2 nM) system with NanoLuc-mNeonGreen calibrator luciferase (2 pM).
