Building Molecular Gates with Nucleic Acid Secondary Structures

Biological systems, at their foundations, use mechanisms that resemble Boolean functions. ON-OFF molecular switches, dose-response actuators, or concentration-dependent biomolecular complex formation behave according to signal thresholds that can be interpreted in terms of ones and zeros, similarly to binary or logic systems. In this thesis, the idea of using triple-helical nucleic acid secondary structures, i.e. triplexes, as biomolecular gates for encoding logic operators was explored. Such RNA-DNA hybrid triplexes were used as modulators of transcription, inducing lower or higher transcription kinetics in a model bacterial system. Therefore, duplex DNA, i.e. the transcription unit, acted as the gate, RNA triplex forming oligonucleotides (TFOs) acted as inputs, and the transcription product was used as output. In addition, by using the specific fluorogenic RNA aptamer Broccoli (i.e., an RNA sequence that binds to a weakly fluorescent ligand and increases its quantum yield), fluorescence emission changes were used as readout of the triplex-based biomolecular gates. Three gates were realized: The complete set AND, OR, and NOT. Triplex formation was characterized by electrophoretic mobility shift assay (EMSA), and temperature-dependent melting point analysis, while transcription modulation was tested in vitro using commercial E. coli s70 -saturated RNA polymerase and designed transcription units. This work adds a new approach to the field of biocomputing, realizing a system that could work in cells in the larger research area of Synthetic Biology.