Development of photo-responsive synthetic RNA devices

The different functions of RNA have led to great and increasing interest in the field of synthetic biology. One such example of versatile RNA molecules used in synthetic biology is aptamers. Aptamers are able to bind to a wide range of target molecules with a high affinity and specificity, which is why they are often compared to antibodies. Using the SELEX method, they can be generated in vitro against target molecules for a broad range of possible applications. In nature, they work as the sensing domain of riboswitches and regulate gene expression by binding their cognate target molecule. Riboswitches can also be generated synthetically from in vitro generated aptamers. In the course of this work, a novel RNA aptamer which selectively binds to one of two light-induced isoforms of a specific small molecule ligand (azoCm) was developed. The potential function of such an aptamer as a riboswitch was evaluated. A previously carried out SELEX experiment against azoCm yielded a specifically binding aptamer (aptamer 42). At the beginning of this work, its secondary structure was analyzed using in-line probing. As it was previously shown aptamer 42 could not work as a riboswitch regulating GFP expression in the model organisms Saccharomyces cerevisiae, other aptamers from the same SELEX were tested under the same conditions using in vivo screening. As no functional riboswitch could be identified, a new SELEX experiment using a new aptamer library was started. The new affinity SELEX yielded aptamers specifically binding to azoCm. However, in vivo screening of aptamers from this SELEX did not result in an azoCm dependent riboswitch. Therefore, a novel light SELEX method was developed and established. This light SELEX protocol enriched isoform selective aptamers by a light-induced conformational change of azoCm during the SELEX process. As in vivo screening of light SELEX aptamers did not yield a riboswitch again, in vitro binding studies of aptamers from the light SELEX were performed. Comparing three aptamers which showed the highest discrimination between the two isoforms of azoCm to each other led to the discovery of a 13 nt sequence motif that only these aptamers shared. A next-generation sequencing experiment performed with the SELEX and light SELEX rounds revealed that this sequence motif (“light motif”) was specifically enriched during light SELEX rounds, but was gradually depleted during the later, more stringent affinity SELEX. Based on this discovery, a new library for SELEX was designed, containing the partially randomized light motif, as well as entirely randomized flanking regions. SELEX performed with this light motif doped library led to a fast enrichment within five rounds, and aptamers from this SELEX were tested regarding their in vivo functionality. From the aptamers tested in vivo, four showed gene regulatory function, however with a low regulatory factor of 1.25- to 1.35-fold. Based on the best functional aptamer B2, partially randomized aptamer libraries were generated for further in vivo screenings. While the regulatory factor of B2 could not be improved using this approach, a modified version of it called B2-1 could be shown to regulate gene expression in yeast cells in an azoCm dose-dependent manner. To learn more about the aptamer B2-1, its binding affinity was analyzed using isothermal calorimetry. The kD of B2-1 was determined to be 23 nM, depending on the calculation model used. A truncated version of B2-1 showed a low micromolar binding to azoCm, indicating that structurally relevant parts of B2-1 had been deleted during the truncation. However, both B2-1 and its truncated version did selectively bind to only one isoform of azoCm, while binding to the other isoform could not be detected. The aptamer developed in this work shows a much stronger discrimination between the two isoforms of its light-switchable ligand than previously reported isoform-selective aptamers. It also shows the highest binding affinity to its ligand compared to the isoform-selective aptamers in literature to date. While a riboswitch based on this aptamer shows only slight regulatory function, dose dependent regulation of gene expression could be shown. This work therefore constitutes the first steps towards the generation of a light dependent riboswitch