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Material and Methods

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. 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.

Results

The results have shown that the fluorescence of the designed Malachite Green is dependend on the malachite green dye. Under conditions containing DNA, T7 RNA Polymerase and malachite green dye, we were able to observe an increase of fluorescence during in vitro transcription(Fig. 1).

Fig.1. 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 vitrotranscription. 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.

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