Project Details
Projekt
Functional and structural comparison of a doxycycline aptamer and two doxycycline riboswitches with different regulatory efficiency and their application in synthetic biology
Subject Area
Biochemistry
Term
since 2025
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 559105806
Synthetic riboswitches are important building blocks in the synthetic biology toolbox. The binding domains of these riboswitches are RNA aptamers obtained by in vitro selection (SELEX). In this application we aim to compare three aptamers that bind doxycycline. However, only two of them are suitable for the construction of riboswitches. The three aptamers resulted from two parallel selections, with one selection specifically favoring the accumulation of structure-switching aptamers. This property appears to be necessary for the construction of riboswitches. We now want to characterize the three doxycycline-binding aptamers comparatively in terms of molecular biology, biochemistry and structure. By comparing the different aptamers, we hope to gain a deeper understanding of the determinants for regulatory activity. Based on these findings, we want to further optimize the aptamers and use them as efficient switching elements in vivo. The switching behavior of the candidate G12 is already far better than all comparable riboswitches published to date. In addition to their importance for applications in chemical biology, the three doxycycline aptamers are also interesting due to their molecular recognition properties. They are not only able to bind doxycycline with high affinity, but also discriminate against tetracycline, which differs from doxycycline only in the position of a hydroxyl group. These aptamers thus form the counterpart to an already known tetracycline aptamer, which has exactly the opposite binding specificity. The structural characterization of these aptamers should therefore provide new insights into the structural basis of the ligand specificity of RNA.
DFG Programme
Research Grants
