Difference between revisions of "Part:BBa K1614016"

Latest revision as of 14:25, 21 September 2015

Hammerhead Ribozyme and Malchite Green Aptamer in BBF RFC 110 transcription cassette

Hammerhead Ribozyme (HHR) and Malchite Green (MG) Aptamer in BBF RFC 110 transcription cassette for live tracking of in vitro transcription.

Usage and Biology

Usage

This BioBrick contains a T7 promoter, a Hammerhead Ribozyme (HHR), a Malachite Green Aptamer and a Hepatitis Delta Virus (HDV) Ribozyme that are connected to each other. This construct is designed so that any RNA insert of interest (ROI) can be cloned in front of the HHR. The HHR is needed to cleave the RNA of Interest (ROI) with a defined 3´ end. Additionally, the Malachite Green Aptamer is expressed and cleaved by the HDV ribozyme. The two RNA products can be purified by denaturing polyacrylamide gel electrophoresis (1A).
To the cleavage side we added a Malachite Green Aptamer. The ligand dependend aptamer folds correctly during the transcription thereby emitting light that was measured in a 384 well plate format.This Aptamer is fluorescent in presence of malachite green dye at 650 nm if excited at 632 nm. This tool was applied to detect the full extension of RNA during in vitro transcription.
This construct was used with the BBa_K1614014 to sense the ATP consumption of in vitro transcription. Experiments have shown that the Malachite Green Aptamer can be used as a universal read out system by adding this Hammerhead Ribozyme that cleaves at the 3’ end of the RNA of Interest. This setup allows the cleavage of an RNA of Interest (ROI) from the Hammerhead Ribozyme-Malachite Green Aptamer (Fig. 1).
Experiments with the Substrate of the RNA cleaving DNAzyme have shown that the Hammerhead Ribozyme-Malachite Green Aptamer cleaves from the substrate. Hence, the substrate and the Hammerhead Ribozyme-Malachite Green Aptamer could be purified on a denaturing polyacrylamide gel electrophoresis (Fig. 2).
We were even able to perform titration curves to enable quantity prediction of transcribed RNA (Fig.3).

Biology

The Hammerhead Ribozyme (HHR) has been employed by many laboratories since its discovery in 1986 (Prody, 1986 and Forster, 1987). HHRs are prominent for their capability of self-cleavage. A HHR consists of three stems of which one is open. The complementary region of this open stem can be designed so that the HHR cleaves off a customized sequence without leaving a scar (Meyer, 2014).

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 vitrotranscription reactions were analyzed using denaturing acrylamide gels.

Fig.2. 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.

Fig. 3.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.

Sequence and Features