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The specific monitoring of small molecules in biochemical reactions is problem that scientist tried to solve since many years. In this project we developed small molecule sensor (SMS) enabling us to analyze biochemical reactions in real-time using a fluorescent readout. Our implemented JAWS software generates us the best ATP-binding Aptamer which is fused to the Spinach Aptamer. In presence of ATP fluorescence will be emitted. This tool enables the sensing of small molecules like ATP that are part of biochemical reactions.

Major Achievements:

• Validation of the JAWS Software
• Sensing of ATP concentrations in biochemical reactions
• Establishment of a dual-fluorescent read-out system to sense small molecules in real-time
• Detailed monitoring of the ATP consumption as well as RNA strand synthesis

The specific monitoring of small molecules in biochemical reactions is problem that scientist tried to solve since many years. In this project we developed small molecule sensor (SMS) enabling us to analyze biochemical reactions in real-time using a fluorescent readout. Using this innovative method, we were able to analyze classical in vitro transcriptions in detail by monitoring the ATP consumption as well as RNA strand synthesis simultaneously. Using a Spinach RNA Aptamer fused to an ATP-binding Aptamer RNA we can specifically sense ATP concentrations in real-time. Our implemented JAWS software generates us the best ATP-binding Aptamer. Thus we can even detect small changes in the concentration of ATP during RNA synthesis. To validate the JAWS software as well as to show the general feasibility of our fluorescent tool-box system we analyzed transcription efficiencies of different RNA polymerases, the influence of the buffer as well as the effect of inhibitors like heparin on transcription. Finally, the combination of the JAWS Software and the fluorescent readout enables the scientific community the possibility to target specifically any small molecule of interest in vivo and in vitro.

Small molecules are known to regulate many cellular functions. Hence, the development of innovative techniques to analyze metabolic pathways became an important field in research. Those assays require a variety of tools allowing the user to detect small molecules even within live cellsFernandez-Suarez2008Tyagi2009. Such methods hold promise to solve the mechanisms of transcription, translation, localization and the function of non-coding RNA. The most common ways to analyze small molecules or cellular pathways include protein-based methods like GFP or molecule reactive probes which have been engineered in the past. Having a fluorescent readout seems to be a valuable implement to provide time-resolved information in vivo and in vitro. Recently, Paige et al. discovered an RNA that mimics the green fluorescent protein (GFP) so called “Spinach” Paige2011Strack2013Strack2015. This Spinach aptamer was generated by systematic evolution of ligands by exponential enrichment (SELEX). In presence of the 3’5-difluoro-4-hydroxybenzylidne imidazolinone dye (DFHBI), RNA forms a stable Spinach-DFHBI RNA aptamer-complex, which is fluorescent. Since then Spinach has been successfully applied by several laboratories to image RNA in live cellsBuxbaum2015Dean2014. Moreover, this RNA has been used as a tool to monitor RNA synthesis in real-timeHöfer2013Pothoulakis in vitro or to sense different small molecule levels in vivoKellenberger2015Kellenberger2013. A prominent example is the sensing of ci-di-GMP concentrations in live cells. To do so, Kellenberger et al. attached a ci-di-GMP aptamer to the Spinach aptamer. In presence of a small molecule (c-di-GMP) the aptamer forms a functional stem which results in the formation of a fluorescent Spinach-DFHBI RNA aptamer-complex. Thus, small molecule concentrations can be determined by a fluorescence read-out system.

In our project we are interested in small molecules that are difficult to sense using common techniques. Here we will describe an innovative system that uses the Spinach2 fused to a specific aptamer to detect small molecules. In 2013 the Jaffrey Lab developed Spinach2 which shows in comparison to Spinach a better folding efficiency and thermostability. Strack2013. To generate aptamers that specifically bind to a small molecule, we will use our software JAWS. Using this set up we will be able to show that our software is capable to support time consuming methods like SELEX, to identify Aptamers that bind specifically to small molecules. As an interesting target, we will sense the small molecule adenosine triphosphate (ATP) in biochemical reactions. A common method that is performed in thousands of laboratories is a in vitro transcriptions To study the function of ribonucleic acids, RNA is generally prepared by in vitro transcriptionBeckert2011. Using bacteriophage DNA dependent RNA polymerases (T7, T3, Sp6), a variety of different RNAs can be enzymatically synthesized in the lab. In this context we want to establish a new biochemical readout method, called real-time SMS, to record simultaneously small molecules (ATP) and enzymatic kinetics (RNA polymerase) using Spinach2-ATP-Aptamer system.

Common techniques to sense small molecules in biochemical reactions are most likely connected to radioactive labeling. The exposure to radioactive sources is known to result in damage of the genetic information and is therefore more and more banished from lab work. However because of its sensitivity, radioactive chemicals are needed to monitor small changes of those molecules.

A common method that is performed in many laboratories is an in vitro transcription (Fig. 1A). This method is usually performed in a black-box manner. To analyze the decrease of nucleotide trisphosphate over time as well as to determine the success of the in vitro transcription, scientists use radioactive labelled nucleotide triphosphates and perform time demanding acrylamide gel electrophoresis.

Fig. 1. in vitro transcription and fluorescent RNA Aptamers.

A

A DNA template is transcribed by and RNA Polymerase (RNAP) into RNA. Tools to analyze the kinetic of the polymerase or consumption of the nucleotide triphosphates during the reaction in real-time are still missing.

B

Secondary structure of the Spinach Aptamer. The Spinach RNA consists of three stem loops that are important for the binding of the dye DFHBI. In presence of the dye a fluoresced signal is emitted.

C

Secondary structure of the Malachite Green Aptamer. In presence of malachite green dye and the Aptamer fluorescence is emitted.

To establish such tool box, two different fluorescent RNA constructs will be applied: The first construct is a fusion of an ATP aptamerSassanfar1993 and Spinach2Strack2013 (Fig. 1B), which we will call Spinach2-ATP-Aptamer system . To improve the binding of the ATP aptamer to ATP we apply our own implemented JAWS software. Using our software, nucleotides which form the stem region of the ATP aptamer can be predicted, which will improve binding properties of this RNA to ATP. The fusion of this aptamer to the Spinach2 enables us a fluorescent read out in real-time. Thus, in presence of this small molecule, the ATP aptamer will form a tight stem loop, which results in a new structural conformation of the Spinach aptamer. Finally after binding of the ATP, the Spinach2-ATP-Aptamer is able to interact with the DFHBI. Hereby fluorescence can be measured at 500 nm if the Spinach was excited at 460 nm. We will generate using our JAWS software different Spinach2-ATP-Aptamers. To validate the software as well as to identify the best Spinach2-ATP-Aptamer system, we will analyze them concerning their fluorescent turn-on effect in presence of ATP.

Fig. 2. Small molecule sensing in real time (Real time SMS).

General overview of our real time SMS tool-box, which allows the monitoring of the ATP consumption during in vitro transcription reaction. For this purpose we designed a DNA template containing a promoter (T7) according to the applied RNA polymerase, RNA of interest (ROI), Hammerhead ribozyme (HHR) and Malachite Green Aptamer (MGA). If the Malachite Green Aptamer is transcribed, an increase in fluorescence can be monitored. The second part of the toolbox is a fusion of Spinach2 to an ATP-binding Aptamer, predicted by our JAWS software. In presence of ATP, the ATP Aptamer forms a stem, enabling the DFHBI dye to bind to the Spinach Aptamer thereby triggering fluorescence emission.

The second important part of the toolbox is a DNA template containing a promoter according to the applied RNA polymerase and a Malachite Green Aptamer (Fig. 1C), which is excited at 630 nm and emits at 652 nm in presents of malachite green dye. If the Malachite Green Aptamer is transcribed, an increase in fluorescence can be monitored. The second part of the toolbox allows a direct analysis of the success of the in vitro transcription in real-time. In an advanced set up, this system can be extended by DNA template encoding for the RNA of interest (ROI) and a hammerhead ribozyme (HHR). Both fragments are inserted between the promotor and the Malachite Green Aptamer. 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 induced by the Malachite Green Aptamer can be applied to compare efficiencies of different RNAs at the same time.

The combination of two aptamers that are able to turn on fluorescence by binding to DFHBI or malachite green provide us the possibility to monitor simultaneously the ATP consumption as well as RNA strand synthesis during in vitro transcription in real-time (Fig. 2). The application of the JAWS software generates us the best ATP-aptamer to identify even small changes during RNA synthesis.

To validate the JAWS-generated aptamers as well as to show the functionality of the Spinach2-ATP-Aptamer system we will apply different bacteriophage RNA polymerases (T7, Sp6, T3) that are commonly used. In addition inhibitors (Heparin) of polymerases as well as the effect of different buffer compositions will be analyzed by our approach.

RNA-Spinach2-Template preparation

Spinach2-DNA templates for in vitro transcription were amplified by extension-PCR and purified using Quiaquick PCR Purification Kit or ethanol precipitation. in vitro transcription was performed in presence of 40 mM Tris-HCl pH 8.1, 1 mM spermidine, 20 mM MgCl2, 0.01% Triton-X100, 4 mM each NTP, 10 mM DTT, 5 % DMSO and 0.1 mg/mL T7 RNA Polymerase. DNA was removed by 1 U/ µL DNase I digest. RNA was purified by denaturing PAGE and eluted in 0.3 M NaOAc pH 5.5. RNA was isopropanol precipitated and concentration determined by NanoDrop measurements.

Purified RNA was renatured in 40 mM HEPES KOH pH 7.5, 125 mM KCl and 3 mM MgCl2 and 20 % RNA. Proper folding was achieved by heating up the RNA to 95 °C for 3 min and then letting it cool down to room temperature.

Functionality of the renatured Spinach2 was determined using 500 nM renatured RNA, 1 mM of ligand (e.g. ATP) 100 µM DFHBI (Lucerna) and 0.2 U/µL of Ribolock RNase Inhibitor (Thermo Scientific). The fluorescence spectrum was measured using a spectro fluorometer (JASCO). Then following settings were applied to measure Spinach 2 fluorescence: Ex= 460 nm and Em=475-600 nm, high sensitivity and 37 °C.

Thin Layer Chromatography to Analyze ATP Consumption During in vitro Transcription

Thin layer chromatography will be applied to analyze ATP consumption during in vitro transcription. To analyze ATP consumptions during the RNA transcription, ATP is converted to AMP after the transcription process by apyrase. in vitro transcription was performed in presence of 40 mM Tris-HCl pH 8.1, 1 mM spermidine, 20 mM MgCl2, 0.01% Triton-X100, 4 mM each NTP, 10 mM DTT, 5 % DMSO and 0.1 mg/mL T7 RNA Polymerase. As DNA template we used the ATP AptamerJAWS1 Spinach2. 5 µL samples were taken every 5 min from the reaction and heated up to 95 °C for 10 min to inactivate the T7 RNA polymerase. To convert ATP to AMP 0.01 U Apyrase and 4 mM CaCl2 were added to each sample and incubated for 1 h at 30 °C. 2 µL of each sample was spotted on a fluorophore coated TLC Plate (ALUGRAM®Xtra SIL G/UV254, Macherey Nagel). TLC was run 4 h 45 min 4:6 Ammonium acetat: ethanol and analyzed with a UV lamp.

Malachite Green Aptamer Template Preparation for in vitro Transcription

Malachite Green Aptamer template (10 µM) was hybridized by heating up a forward and reverse oligo to 95 °C. DNA was cooled down to room temperature. dsDNA was then stored at -20 °C.

Malachite Green Aptamer Activity

Malachite Green Aptamer activity was tested by transcribing the prepared DNA-Template in vitro with T7 polymerase as described above and in presence of 1 mM malachite green (Sigma). To ensure synchronic initiation a master mix containing the buffer, enzymes, dye and template were pipetted separately. The assay was performed in 384 well micro titer plates (black, flat round, transparent bottom [Corning, 3540]) on a 20 µL scale. Evaporation during transcription was prevented by using a sealing tape (#232701, Nunc). Measurements on micro titer plates were performed in a microplate reader (Tecan Safire 2). Following parameters were chosen for the assay setup: Ex= 630 nm and Em=652 nm, 10 nm excitation/ emission bandwidth, high sensitivity flash mode and 40 µs integration time. The reaction was measured every 30 sec at 37 °C. To ensure the functionality of the assay, samples of the in vitro transcription were analyzed on a 10 % 8 M urea PAGE.

Time-resolved in vitro Transcription

To describe in vitro transcription time-resolved in terms of NTP consumption, following conditions were applied: 500 nM renatured ATP Aptamer Spinach2 RNA, 10 µM Malachite Green Aptamer DNA template, 40 mM Tris pH 8.1, 1 mM spermidine, 20 mM MgCl2, 0.01% Triton X-100, 4 mM each NTP, 10 mM DTT, 5 % DMSO, 100 µM DFHBI, 1 mM malachite green, 0.1 mg/mL T7 RNA Polymerase, 0.2 U/µL Ribolock RNase and 0.1 U Pyrophosphatase (Thermo Scientific). The assay was performed in 384 well micro titer plates (black, flat round, transparent bottom [Corning, 3540]) on a 20 µL scale. Evaporation during transcription was prevented by using a sealing tape (#232701, Nunc). Measurements on micro titer plates were performed in a microplate reader (Tecan Safire 2).

Influence of Inhibitors on in vitro Transcription

To analyze the influence of inhibitors on the in vitro transcription, heparin (0.7 mg/mL and 1.3 mg/mL) was applied. in vitro transcriptions were performed similar to “time-resolved in vitro Transcription” as described above.

Quantification of RNA Concentrations using Malachite Green Aptamer Fluorescence Read-Out

The T7-Malachite Green DNA template was applied for in vitro transcription as described above. RNA was purified by denaturing PAGE, eluted and recovered by NaOAc/isopropanol precipitation. The RNA was purified from remaining salts using Amicon Ultra-0.5 mL centrifugal filters 3K (Merck Millipore). For preparation of a calibration curve, RNA solutions of different concentrations were refolded (95°C 3min, in 1x renaturing buffer) 7. After addition of 100 µM DFHBI, 100 µM malachite green, 10 mM DTT, 4 mM ATP, 4 mM GTP, 4mM CTP, 4 mM UTP, 1x transcription buffer (1mM spermidine), 0.0017 U pyrophosphatase, 0.46 U Ribolock, 0.075 % glycerol fluorescence, fluorescence was measured on micro titer plates using microplate reader (Tecan Safire 2). Following parameters were chosen for the assay setup: Ex= 630 nm and Em=652 nm, 10 nm excitation/ emission bandwidth, high sensitivity flash mode and 40 µs integration time. The reaction was measured every 30 sec at 37 °C.

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

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.

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.