B4 - Nucleic acid-based molecular automatons: logic gating by dynamic transcriptional control in vitro and in cellulo and underlying molecular mechanisms

We have developed a rotaxane-based DNA nanomachine that potentially self-regulates (m)RNA transcription. This linear actuator autonomously switches between dynamic and static states in a self-repeating seesaw motion. It uses T7 RNA polymerase (T7RNAP) to propel a macrocycle along its axis, with transcription initiation depending on T7 promoter sequence and geometry, which is controlled by macrocycle hybridization. The system self-regulates in that a new transcriptional cycle only begins when the previous cycle is complete. Therefore, the actuator has the potential to control the presence and amount of (m)RNA molecules encoded in its axis and thus represents a self-regulatory system for dynamic transcriptional control. Our proposal aims to expand upon this design to create versatile actuators capable of multi-layered, logic-gated transcription cascades, responding to various input signals. Initially, we will focus on developing different AND- and XORgates, triggered by the presence or absence of specific combinations of DNA or RNA input signals. Their outputs will be short RNAs that serve as inputs to trigger the next adjacent gate. The various gates then will subsequently be used to construct 3-bit adders, whose outputs will be detected by the production of fluorescent proteins, molecular beacons (MB), or fluorescent RNA-aptamers such as broccoli, corn, or pepper. Fluorescence will be our main method to study the dynamics of the complexes that constitute each logic gate. Thus, the 3-bit adder consists of logic gates based on dynamic nucleic acid complexes in which DNA macrocycles are arranged on a 6-helix bundle DNA origami to ensure dynamic transcriptional control of T7RNAP. To achieve orthogonality on the polymerase side, a further aim is the use of LOV-T7RNAP that can be triggered by light/dark, and also the in vivo evolution of T7RNAP mutants that utilize different promoter sequences than those of T7RNAP. For the latter, we will use the synthetic orthogonal replication system that enables accelerated evolution in E. coli, established this year by Jason Chin. A further trigger for the dynamics of the system can be the cis/trans-isomerization of azobenzene-containing input oligos. The interplay of these RNAP variants and trigger signals controls the dynamics of the overall system by transcribing different RNA outputs that are used as input for each logic gate. They will allow for a significantly greater bandwidth of input and output signals, which in turn would increase the complexity and accuracy of the mathematical operations, as well as the variety of the final output events. A goal of longer term is to apply these, or modified 3-bit adders in bacteria. However, it is currently completely unknown whether and how the DNA nanomachine can be used in cellular systems for artificially controllable autoregulation of functional RNAs (mRNA, ncRNAs). Thus, another main goal of this proposal is to set the stage for intracellular application of these DNA automatons to not only control the level of specific functional RNA molecules in cells, but also to employ them for intracellular logic gating. To this end, we will systematically examine various macrocycles for promoter dynamics, structure and integrity, transcriptional efficiency and turnover - first in vitro and then in a simple cellular host system. Ultimately, the actuator will be converted into a self-regulating artificial mRNA expression system with integrated and logic-gated transcriptional control and established as an automaton that controls the expression of specific mRNAs (and their translation products) in cells. The fully synthetic system described here could have broad application in synthetic biology, engineered bacteria, yeast, or other cultured cells used for biotechnological production of desired products when self-directed regulation of a particular RNA is desired.