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Results

Establishment of a System to Sense Small Molecule Using the Spinach2 Aptamer

Fig. 3. Fusion of Aptamers to Spinach to generate fluorescent small molecule sensors.
A

For the design of a new small molecule sensor, the second stem of the Spinach2 aptamer can be exchanged by an aptamer that binds specifically to a small molecule. We fused an ATP-binding Aptamer (yellow) to the Spinach.

B

We applied our JAWS software to predict the best ATP-Aptamers, that can form the best stem structure (blue and red highlighted) in presence of ATP.

C

The JAWS predicted stems can be fused to the Spinach Aptamer. The software can be validated by analyzing the fluorescence emission in presence of ATP and DFHBI (Fig.4B and C).

To establish a stable, reproducible system in lab that is able to sense small molecules in real time using a fluorescent readout, we applied the already published c-di-GMP Spinach2 system firstKellenberger2013. To design new small molecule sensors, we adapted the system described by Kellenberger et al. We exchanged the second stem of Spinach2 by three different ATP Aptamer systems (Fig. 3). All of them are based on an ATP Aptamer described by Szostak. ATP Aptamer Spinach2 is the original aptamer that was generated by Szostak. ATP AptamerJAWS1 Spinach2 and the ATP AptamerJAWS2 Spinach2 contain the core sequence of the original ATP Aptamer, but have a modified stem, generated by our software JAWS. In presence of the ATP ligand, the ATP aptamer should change its tertiary structure and forms together with the Spinach2 a stem. The stem formations should finally result in an increase of the fluorescence. Using this read-out system we can compare the JAWS-generated Aptamers with the original one, described by Szostak. If our JAWS software works nicely, we should get a better fluorescent read-out with ATP AptamerJAWS1 Spinach2 or ATP AptamerJAWS2 Spinach2 in comparison to the original ATP Aptamer Spinach2 construct.

Fig. 4. Establishment of a system to sense small molecule using the Spinach2 Aptamer.
A

Emission spectrum of the original Spinach2 Aptamer, which was applied as an internal control.

B

As another internal control, we reproduce the data for the c-di-GMP Spinach2 system, published by Kellenberger et al.. Indeed, highest fluorescence maximum for the c-di-GMP Spinach2 system was measured in presence of the ligand.

C

Analysis of the fluorescent properties of our ATP Aptamer Spinach2 constructs. The Spinach2 containing the Szostak ATP Aptamer shows the lowest fluorescence of all three ATP Aptamer Spinach2 variations. The JAWS-generated ATP AptamerJAWS1 Spinach2 and the ATP AptamerJAWS2 Spinach2 show higher fluorescence maxima in presence of ATP.

To analyze the functionality of our small molecule sensor system, we amplified all constructs, including, Spinach2, c-di-GMP Spinach2 and ATP Aptamer Spinach2 sensors by PCR and transcribed them in vitro successfully. The RNA was analyzed for its specific binding to a small molecule by a highly sensitive fluorometer. Figure 4 shows the results of the fluorescence measurements. All Spinach2 systems show fluorescence maxima at 500 nm in presence of the ligand. The original Spinach2Paige2011 was applied as an internal control and shows the highest fluorescence intensity (Fig. 4A). As another internal control we tried to reproduce the data for the c-di-GMP Spinach2 system (Fig. 4B). Indeed, highest fluorescence maximum for the c-di-GMP Spinach2 system was measured in presence of the ligand. Thus we were able to reproduce the data shown by Kellenberger et al. In addition, we observed a new second peak for the c-di-GMP Spinach2. The second maxima appears at Em= 552 nm. The red light shift also exists in the spectrum of the c-di-GMP Spinach2 without ligand with a lower peak. Finally, we analyzed the fluorescent properties of our new ATP Aptamer Spinach2 constructs (Fig. 4C). The Spinach2 containing the Szostak ATP Aptamer shows the lowest fluorescence of all three ATP Aptamer Spinach2 variations. The JAWS-generated ATP AptamerJAWS1 Spinach2 and the ATP AptamerJAWS2 Spinach2 show higher fluorescence maxima in presence of the ligand. Due to its perfect fluorescence properties we applied the ATP AptamerJWAS1 for further studies.

Common Techniques of Sensing Small Molecules in Biochemical Reactions

Fig. 5. Analysis of an in vitro transcription by thin layer chromatography.

ATP consumption during the transcription was monitored by TLC. ATP was converted to AMP by an apyrase reaction in order to separate it from other NTPs. Using TLC the consumption of ATP by the polymerase cannot be monitored properly.

Analyzing biochemical reactions depend on radioactive labeling. Yet, many laboratories try to avoid the usage of radioactivity since it is connected to affect the health. Our established ATP Aptamer Spinach2 sensor system enables the sensing low concentrated small molecules like ATP within in vitro assays. In this context the change of ATP concentration during the in vitro transcription reaction is an attractive target to study. NTPs are genially consumed by a RNA polymerase over time. To monitor the decrease of ATP during a successful in vitro transcription, we applied thin layer chromatography first (Fig. 5). To analyze ATP and not GTP/CTP or UTP by the TLC, the remaining ATP was converted to AMP via an apyrase reaction. However, we cannot identify significant changes of the ATP concentration on the TLC plate because of the low sensitivity of this method.

Highly Sensitive Sensing of Small Molecules via Spinach in Biochemical Reactions in Real time

Fig. 6. Real time monitoring of the RNA synthesis using a Malachite Green Aptamer.
A

To analyze RNA synthesis in real time we applied a DNA template encoding for the RNA of interest (ROI) and a hammerhead ribozyme (HHR). Both fragments are inserted between the promotor (T7) and the Malachite Green Aptamer (MGA). Inducing the hammerhead ribozyme allows cleavage during the in vitro transcription. Hereby two RNA fragments emerge, the ROI and the hammerhead ribozyme fused to Malachite Green Aptamer (HHR-MGA). Using such setup, the emitted fluorescence of the HHR-MGA will be independent on the applied ROI. Thus the fluorescence is induced by the Malachite Green Aptamer that can be applied to compare efficiencies of different RNAs at the same time. B

As a proof of principle we performed transcriptions and monitored the malachite green fluorescence signal in real time.

C

To confirm the results of the fluorescence measurements as well as the transcription efficiency is not hampered by the malachite green dye, all in vitro transcription reactions were analyzed using denaturing acrylamide gels.

Common techniques like TLC have a very poor limit of detection. Especially in our studies, a highly sensitive method is needed to visualize ATP concentration in biochemical reactions. Thus, we can apply our ATP AptamerJAWS1 Spinach2 system so sense consumption of ATP in real time (Fig. 6). To show the general feasibility of our system we titrated the T7 RNA polymerase concentration in in vitro transcription reaction. We monitored the fluorescence signal of the ATP AptamerJAWS1 Spinach2 in regular intervals. In presence of a high RNA polymerase concentration we would expect the highest turn-off effect of the fluorescence. In Figure 6B we show the results of the real time measurements of the spinach fluorescence. As expected we can show that the ATP signal decreases rapidly in presence of a high concentration of polymerase. In contrast, if less RNA polymerase was applied, the measured fluorescence is much higher. To confirm the results of the fluorescence measurements, we analyzed the in vitro transcription reaction using denaturing acrylamide gels in addition (Fig. 6C). Similar to the fluorescent readout, we were able to identify the most intense bands in presence of high T7 RNA polymerase.

in vitro Transcription Using a Fluorescent RNA of Interest

To substitute the time-consuming analysis of in vitro transcription by denaturing acrylamide gel electrophoresis, we established a second read-out system. Using this system we want to monitor RNA synthesis in real time (Fig. 7). For this purpose we applied a DNA transcription template which encodes the Malachite Green AptamerBaugh2000. This aptamer binds to the malachite green dye and differs theoretically from the ATP Aptamer Spinach2 in its excitation and emission spectra.

Fig. 7. Real time monitoring of the RNA synthesis using a Malachite Green Aptamer.
A

To analyze RNA synthesis in real time we applied a DNA template encoding for the RNA of interest (ROI) and a hammerhead ribozyme (HHR). Both fragments are inserted between the promotor (T7) and the Malachite Green Aptamer (MGA). Inducing the hammerhead ribozyme allows cleavage during the in vitro transcription. Hereby two RNA fragments emerge, the ROI and the hammerhead ribozyme fused to Malachite Green Aptamer (HHR-MGA). Using such setup, the emitted fluorescence of the HHR-MGA will be independent on the applied ROI. Thus the fluorescence is induced by the Malachite Green Aptamer that can be applied to compare efficiencies of different RNAs at the same time. B

As a proof of principle we performed transcriptions and monitored the malachite green fluorescence signal in real time.

C

To confirm the results of the fluorescence measurements as well as the transcription efficiency is not hampered by the malachite green dye, all in vitro transcription reactions were analyzed using denaturing acrylamide gels.

To analyze the functionality and fluorescent properties of the Malachite Green Aptamer we performed in vitro transcriptions and measured fluorescence in regular intervals. We prepared two negative controls (Blank) which were either depleted with T7 RNA Polymerase or without malachite green. The functional setup contains DNA template, polymerase as well as malachite green dye to monitor the transcription in real time by an increase of the fluorescence signal. If our Malachite-Green Aptamer read-out system functions properly, we expect an increase at an emission wavelength of 650 nm only for functional setup. The blank values should stay at zero. The results (Fig. 7) indicate that we do not see any increase of fluorescence for the blanks. All measured values show a fluctuation around 0. The graph of the functional aptamer with malachite green and T7 polymerase shows an increase of fluorescence over time. We were able to record a classical enzyme kinetic curve of a polymerase using the malachite green system (Fig. 7B). Furthermore, to confirm our data and to show, that the transcription efficiency is not hampered by the malachite green dye, we performed denaturing acrylamide gel electrophoresis (Fig. 7C). The gel shows similar band intensities in presence as well as in absence of the dye if T7 polymerase was added to the reaction. Thus malachite green does not influence our transcription efficiency.

in vitro Transcription recording the Nucleotide Consumption in Correlation to de novo RNA Synthesis

Fig. 8 Simultaneous monitoring of ATP consumption and RNA synthesis during transcription.

in vitro transcription reaction was performed in presence of the ATP AptamerJAWS1 Spinach2/DFHBI and Malachite Green Aptamer DNA template/malachite green dye. Fluorescence was measured in regular intervals. During the reaction we were able to sense the decrease of ATP AptamerJAWS1 Spinach2 fluorescence and an increase of the fluorescence corresponding to the Malachite Green Aptamer. Controls (w/o T7 RNA polymerase) show no change in the fluorescence signal over time. Thus ATP consumption and RNA strand synthesis can be analyzed in real time.

As we were able to establish a highly sensitive fluorescent read out for ATP concentrations as well as for the RNA synthesis, we wanted to combine both methods. We performed in vitro transcription reactions in presence of the ATP AptamerJAWS1 Spinach2/DFHBI and Malachite Green Aptamer DNA template/malachite green dye. Fluorescence was measured in regular intervals. During the reaction we were able to sense the decrease of ATP AptamerJAWS1 Spinach2 fluorescence and an increase of the fluorescence corresponding to the Malachite Green Aptamer (Fig. 8). The results show that the transcription reaction reaches its maximum after 140 min.

Fig. 9. Real time monitoring of RNA synthesis and ATP consumption in presence of inhibitors.
A

Model of the transcription using our Spinach/Malachite Green readout system in presence of a polymerase inhibitor (I) such as heparin. The inhibitor results in a stop of the transcription. B

We determined the influence of the inhibitor heparin on a T7 RNA polymerase transcription. For this purpose we applied our new fluorescent readout system. Fluorescence emission of ATP AptamerJAWS1 Spinach2 and Malachite Green Aptamer is not increasing over time. For the positive controls (w/o heparin) the fluorescence increases as expected over time.

In further studies, we added heparin to the in vitro transcription which is known to be a potent inhibitor of the T7 RNA polymeraseSastry1997. Here we expect a stable level of the ATP signal and reduced fluorescence at 650 nm (Malachite green). In Figure 9 we can show that indeed heparin results in low fluorescence signal and ATP concentrations stay at the same level over time. Thus transcription reaction is inhibited by this molecule.

High Throughput Buffer Test under Low Input Conditions

Fig. 10. Application of the Spinach/Malachite Green readout system to improve buffer conditions for the transcription.

As DNA template we applied the Malachite Green Aptamer with T7 promotor. Using our fluorescent read out system we can determine different transcription efficiencies as well as ATP consumption in real time.

A

In presence of the HEPES buffer no increase of the Malachite Green Aptamer fluorescence signal was monitored. Furthermore the signal of the ATP Aptamer Spinach2 stays also at the same level as the blank.

B

Tris-HCl buffer show a decrease of Spinach2 fluorescence and an increase of the Malachite Green Aptamer signal.

C

The transcription buffer shows the highest slope of Malachite Green Aptamer fluorescence and the fastest decrease of the ATP Aptamer Spinach2 fluorescence.

Adjusting an in vitro transcription to its optimal conditions is usually connected to big efforts. The efficiency and the final RNA yield of each transcription depend on the applied buffer conditions, polymerase as well as DNA template. In our project several RNAs were generated by in vitro transcription. However, we could not achieve high efficiency for some constructs such as the “substrate of RNA-cleaving DNAzyme”. To show the general applicability of our Spinach/Malachite green system to optimize transcription conditions in the shortest time possible, we tested different buffers systems first (Fig. 10). Especially buffers are known to have a huge influence on the activity of the polymerase which can probably improve the transcription of the DNA template “substrate of RNA-cleaving DNAzyme”.

For the buffer screen we applied six different buffer systems: Three transcription buffers pH 8.1 with different spermidine concentrations ranging from 0 mM to 10 mM, Tris-HCl buffer pH 8.1, HEPES buffer pH 7.5 (similar to the renaturing buffer) and a buffer including BSA pH 8.1. All buffers were analyzed using the same conditions as described in previous assays. As DNA template we applied the Malachite Green Aptamer with T7 promotor. Using our fluorescent read out system we can determine different transcription efficiencies. In presence of the HEPES buffer no increase of the Malachite Green Aptamer fluorescence signal was monitored (Fig. 10A). Furthermore the signal of the ATP Aptamer Spinach2 stays also at the same level as the blank. The BSA buffer and the Tris-HCl buffer (Fig. 10B) show a decrease of Spinach2 fluorescence and an increase of the Malachite Green Aptamer signal. The transcription buffer (1 mM and 10 mM) shows the highest slope of Malachite Green Aptamer fluorescence and the fastest decrease of the ATP Aptamer Spinach2 fluorescence (Fig. 10C). Thus we can detect huge differences in the transcription by applying our Spinach/Malachite Green set up. In addition we tested DNA template “substrate of RNA-cleaving DNAzyme”, which was difficult to transcribe (Fig. 11).

Fig.11. Monitoring of the ATP consumption and RNA synthesis of difficult transcription templates.
A

Using our fluorescent tool box we can demonstrate again that substrate of RNA-cleaving DNAzyme causes problems during transcription. We can identify a slight decrease of Spinach fluorescence and increase in the Malachite Green signal. B

We were applying a DNA template containing a HHR in front of the Malachite Green Aptamer, we analyzed cleavage by denaturing acrylamide gel electrophoresis. We could identify the cleaved HHR-MGA RNA by high sensitive Sybr Gold staining.

Using our fluorescent tool box we can demonstrate again that substrate of RNA-cleaving DNAzyme causes problems during transcription. We can identify a slight decrease of Spinach fluorescence and increase in the Malachite Green signal. Thus this RNA yield of this transcription is low (Fig. 11A). As we were applying a DNA template containing a HHR in front of the Malachite Green Aptamer, we analyzed cleavage by denaturing acrylamide gel electrophoresis (Fig 11B). We could identify the cleaved HHR-MGA RNA by high sensitive Sybr Gold staining.

Employing different Polymerases for Universal Setup Conditions

Fig. 12. Determination of the transcription efficiency of different polymerases.
A

We applied our Spinach/Malachitgreen setup to different RNA polymerase such as T7Phi 2.5, T3, Sp6 and E. coli. B

ATP consumption (ATP-AptamerJWAS1 Spinach fluorescence) and RNA synthesis (Malachite Green fluorescence) were measured in real time. ATP was fast consumed and RNA transcribed by T7 and T3 polymerases. SP6 shows activity as well. E. coli RNA polymerase was not showing any activity.

To overcome the dependence of the setup to apply a specific polymerase we designed new transcription templates containing different promoters for a subset of commercial available RNA polymerase such as T7 Phi 2.5, T3, Sp6 and E. coli (T7A1) (Fig. 12A). All polymerases have been tested for 12 hours at 37 °C and were measured in regular time intervals using TECAN Safire. The results show that the decrease of ATP AptamerJWAS1 Spinach2 activity is connected to the speed of the RNA polymerase. The data indicates that the reduction of ATP goes faster for T7 and T7Phi 2.5 than for the other candidates (Fig. 12B). Therefore the fluorescence of ATP AptamerJAWS1 Spinach2 decreases. The T3 polymerase is a little bit slower than the T7 RNA polymerase and does not reach the same bottom point. Nevertheless the graph decreases still over time and the polymerase seem to be active even after 500 min of the reaction. The Sp6 shows activity as well. The Sp6 leads to higher Malachite Green Aptamer production by using less ATP or leading to more functional RNA, while the T3 has a lower ATP Aptamer Spinach2 activity than Malachite Green Aptamer.

Establishment of Titration Curves for Quantification of the in vitro Transcription

Fig. 13. Calculation of RNA yields in real time using the Spinach/Malachite Green system.
A

Malachite Green Aptamer RNA was titrated to different concentrations (10 µM, 7.5 µM, 5 µM, 2.5 µM, 1 µM, 0.5 µM and 0 µM of RNA) and the fluorescence determined using a multiwell-plate reader. A calibration curve was calculated and applied to quantify RNA yields in real time. B

As an example we calculated the RNA amount of the different transcriptions in presence of different DNA template concentrations (0-2.5 nM). As expected best RNA yields were determined in presence of the highest DNA template concentration.

The quantification of fluorescence is necessary to estimate the total amount of RNA synthetized in the in vitro transcription. For this purpose we transcribed the Malachite Green Aptamer and purified it by denaturing PAGE. Finally Malachite Green Aptamer RNA was titrated to different concentrations (10 µM, 7.5 µM, 5 µM, 2.5 µM, 1 µM, 0.5 µM and 0 µM of RNA) and the fluorescence determined using a multiwell-plate reader in presence of the dye and all other transcription components (Fig. 13). We were able to obtain a calibration curve (Fig. 13A) with a correlation coefficient of 0.99 which enables us to calculate the final RNA yields of different in vitro transcription reactions (Fig. 13B). As an example we calculated the RNA amount of the different transcriptions in presence of different DNA template concentrations. As expected best RNA yields were determined in presence of the highest DNA template concentration.