Part:BBa_K1791000:Experience

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Applications of BBa_K1791000

THEOPHYLLINE APTAZYME IN VITRO:

The theophylline-responsive aptazyme used in our project was previously engineered to function in vivo in the yeast species Saccharomyces cerevisiae (Win and Smolke 2007). The device was designed to modulate the cleavage of the host RNA via addition of exogenous theophylline (theophylline is not normally present in yeast cells). Binding of theophylline to the aptazyme is predicted to induce a conformational change in the RNA that promotes a cleavage competent structure. Initially, we wanted to determine if the device was viable under in vitro conditions. To test this, we generated PCR products of the full length aptazyme and used this as a template for in vitro transcription by T7 RNA polymerase (Fig. 1).

Media:Uleth15_Theophylline_AptazymeIVT_Sept_1_2015.jpg

Figure 1. Theophylline Aptazyme in vitro transcription.

We readily detected large amounts of aptazyme in vitro transcript (Fig 1), however we noted that a significant amount of the transcript was truncated and of a length consistent with cleaved aptazyme, even in the absence of theophylline. We hypothesized that the premature cleavage of the aptazyme may have resulted from the temperature conditions in which our in vitro transcription was performed; non-optimal temperatures may impair proper folding of the structure resulting in premature cleavage during in vitro transcription. We therefore tried performing the in vitro transcription at room temperature and 37°C (Fig. 2).

Media:Uleth15_Theophylline_Aptazyme_Sept_2_2015.jpg

Fig 2.Theophylline Aptazyme in vitro transcription at 24°C and 37°C time course.

Interestingly, in the in vitro transcriptions that were carried out at room temperature for one hour, approximately half of the transcripts were full-length, while longer incubations at room temperature or at 37°C showed mostly cleaved aptazyme transcripts. This is the first time the performance of this device has been exhibited in vitro. In an attempt to further improve the yield of full-length aptazyme transcript, we tested a wide variety of temperatures, incubation times, and buffer conditions for in vitro transcription (Fig. 3).

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Figure 3. Theophylline Aptazyme in vitro transcription temperature and magnesium testing.

Unfortunately, none of the in vitro transcription conditions tested were able to generate additional full-length transcript. Next, we wanted to see if addition of theophylline might still result in further cleavage of the aptazyme. Thus, we performed cleavage assays of the partially full-length in vitro transcripts using theophylline concentrations ranging from 100nM to 1mM, and analyzed the products on denaturing polyacrylamide gels (Fig. 4).

Media:Uleth15_Theophylline_Aptazyme_Cleavage_Assay_Purified_in_vitro_Sept_8_2015.jpg

Figure 4. Theophylline Aptazyme Cleavage Assay. Purified in vitro.

Unexpectedly, after incubation at room temperature for 30 minutes, aptazyme transcripts were all cleaved, irrespective of theophylline concentration. This indicates that further optimization of the cleavage conditions must be undertaken to perfect the performance of the theophylline aptazyme in vitro.

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