Cellular measurement and computation with RNA circuits

Summary

RNA stands out as an increasingly attractive molecule for engineering biology, in part due to the programmable nature of RNA:RNA interactions. The Cellular Engineering Group is developing predictable and versatile RNA circuits based on nucleic acid strand displacement reactions, to meet emerging measurement and computation needs in biological systems.

Description

Once considered a passive intermediate, RNA is now implicated in many cellular functions. RNA molecules sense metabolites, regulate gene expression, and organize cellular materials. This has led to a broad interest in harnessing RNA for engineering biology. A key advantage of engineering cellular circuits using RNA, rather than protein-based transcription factor circuits, lies in the programmability of RNA interactions via predictable base pairing rules. RNA-based circuits have a number of other benefits over protein-based circuits: 1) without the need for translation, RNA circuits are less burdensome to the cell and exhibit fast response times, 2) RNA circuit components are often short sequences, reducing their genetic footprint and increasing their genetic stability, 3) myriad RNA sequencing techniques exist that enable unparalleled measurements of system behavior and performance, and 4) because they rely on universal RNA:RNA base pairing interactions, RNA circuits may retain function across diverse organisms.

To enable more predictable and versatile RNA circuits, the Cellular Engineering Group is developing genetically encodable toehold-mediated strand displacement (TMSD) circuits. In these circuits, single-stranded RNA inputs react with partially double-stranded RNA gates to displace output strands. These reactions are facilitated by single-stranded overhangs on the RNA gates, called toeholds. Typically, strand displacement exposes a new toehold on the output RNA, enabling the output to participate in downstream TMSD reactions. TMSD cascades can be programmed via sequence complementarity to execute logic, signal amplification, and complex decision making. The Cellular Engineering Group has recently developed cotranscriptionally encoded RNA strand displacement (ctRSD) circuits, a platform to transcriptionally encode RNA TMSD components. We have validated that ctRSD enables predictable and tunable circuit dynamics in vitro. ctRSD circuits could enable precise, predictable, and scalable RNA circuit engineering. Further, these circuits should enable information processing beyond what is possible with existing transcription-based circuits, such as pattern recognition.

To facilitate RNA circuit engineering, the Group is also developing techniques to measure RNA interactions and RNA circuit dynamics in situ.

 

Figure: (A) Traditional toehold mediated strand displacement where pre-prepared inputs and gates react to produce an output with an exposed toehold. (B) In cotranscriptionally encoded RNA strand displacement (ctRSD) circuits, RNA gates are encoded as hairpins that self-cleave after folding to allow all circuit components to be produced in situ. (C) A fluorescent DNA reporter can measure ctRSD output production (left). The reaction dynamics can be predictably tuned by varying the relative concentrations of the input and gate templates (plot).

Animation: A GIF demonstrating ctRSD circuit operation. Two gates independently fold and self-cleave during transcription. The left gate can then react with an input strand via toehold-mediated strand displacement to produce an output strand that serves as an input to the right gate.

RELATED PUBLICATIONS

Schaffter, S.W., Strychalski, E.A. Cotranscriptionally encoded RNA strand displacement circuits. Science Advances. 2022;8(12). DOI:  https://www.science.org/doi/10.1126/sciadv.abl4354