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
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).
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).
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21Illegal BamHI site found at 85
Illegal BamHI site found at 117
- 23COMPATIBLE WITH RFC
- 25Illegal NgoMIV site found at 124
Illegal NgoMIV site found at 153
- 1000COMPATIBLE WITH RFC