## 4 Discussion  
In this study, we report the design and construction of a novel synthetic transcriptional regulator, SWT, whose transcriptional state is regulated in a switch-like manner by the presence of a cognate RNA trigger. Both natural and synthetic terminators could be converted to SWT by engineering additional toehold domains upstream of the rho-independent terminator sequences. Our best SWT, T500 SWT, showed an extremely low level of leakage and a dynamic range close to the best STAR regulator reported, demonstrating its potential as a promising gene regulatory part. We combined the SWT with THS to create 2-input AND logic gates capable of multilevel regulation both at transcription and translation stages; this multilevel regulator showed leakage levels down to cellular autofluorescence for all logical FALSE states. Further, these multilevel regulators can be easily multiplexed to form 3-input/2-output logic circuits. To test the generality of our design approach, we constructed a multilayered circuit with a TetR repressor under the control of T500 SWT in the first layer and a GFP reporter under the control of TetR in the second layer. We confirmed that the tight leakage control through T500 SWT allowed constructing a functional 2-layer circuit, an improvement over THS and early STAR regulator. Thus, we provide evidence that the SWT can serve as novel transcriptional regulatory elements with desirable dynamics and very low leakage and may find its use in complex synthetic circuits. Among the SWT designs we tested, the best T500 SWT showed tight leakage control comparable to the best STAR regulator reported while demonstrating much less leaky expression as compared to THS elements; however, the expression level in its ON state was lower than other STAR and THS regulators. To further improve expression levels, a number of sequence optimization strategies can be applied to SWT. For the current version of T500 SWT, we combined the toehold binding domain of the best STAR regulator (STAR Target 1) and RBS sequence from the THS library [30,31]. Higher expression levels may be achieved if the sequence contexts of multiple components such as RBS, linker sequences, and toehold domains of SWT are adjusted at the same time for optimal sequence combination. For instance, we also tested two other toehold sequences from the STAR library [33] for T500 SWT and observed that the ON-state expression level was changed while maintaining the same low leakage level in the OFF state (Figure S3). Therefore, it seems likely that the ON-state can be further optimized without disrupting the tight leakage control of SWT. In case a tighter leakage control is desired, alternative approaches to further reduce leakage level may include elongating the terminator stem and engineering the terminator stem to partially sequester RBS [42]. The design flexibility of RNA structures will likely lead to new developments to circumvent these current limitations. Our experiments demonstrated SWT as switchable control elements for transcriptional regulation that can complement STAR transcription regulators and enrich the toolbox of RNA synthetic biology [55]. Due to the working mechanism that terminates transcription of the downstream gene in its off state, the resource burden on the host organisms is minimized while achieving tight leakage control. In a multilayered circuit architecture, a low level of input noise can be quickly amplified, requiring much better control of leaky expression at each regulatory level; SWT can provide the tight regulatory control that meets the stringent requirement of multilayered circuits. In addition, these properties of SWT would be effective at managing the production of toxic molecules and could find use in the development of biocontainment technology [56, 57, 58]. Taken together, SWT, as a new set of modular transcriptional regulatory components, could provide a much-needed toolkit to build complex synthetic circuits and accelerate advancement in RNA synthetic biology [59].