Engineered Bacterial Transcription Units Controlled by RNA:DNA Triplexes

Organisms can control gene expression at different levels, for example by rendering some parts of the genome less accessible to RNA polymerase or by changing the affinity of the transcription complex for specific promoter sequences using transcription factors. Recently, special RNA molecules, called long non-coding RNAs, are more and more at the center of scientific interest due to their involvement in gene regulation. Although experimental evidences are still scarce, it was proposed that they can directly interact with regulatory genome sequences via formation of triplex structures. In this study, a recently proposed model mechanism for gene regulation via triplex formation is further investigated for the production of a molecular gating system reproducing the logic gate XOR, where the triplex forming RNA molecules represent the input, while the transcribed product represents the gate output. To achieve this goal, a bacterial expression unit was designed containing a triplex-controlled promoter positioned upstream of a sequence expressing the fluorogenic aptamer Broccoli. The time-dependent fluorescence signal of Broccoli production was used to estimate the transcription rate while the triplex formation was characterized by gel electrophoresis. The results showed that the input combinations (0-0, 0-1, 1-0, and 1-1) generated outputs in agreement with the designed XOR gate (0, 1, 1, and 0, respectively), demonstrating the feasibility of building triplex-mediated molecular computing units. Furthermore, this molecular gate mechanism could be used to develop a threshold gate, i.e., the fundamental node of neural networks, and thus it can be considered a universal molecular computing mechanism. In conclusion, triplex formation and fluorogenic aptamers can be used to study a new level of gene regulation. This is of central importance in fundamental biology, as well as for the development of molecular computing systems. In addition, this study could be used for the discovery of new targets against harmful bacteria and thus for increasing the availability of antibacterial molecules.