RNA computation and metrology for engineering biology

Summary

RNA is 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 molecular computation and measurement 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 cascades of protein transcription factors, 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 can be 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 techniques, many involving RNA sequencing, exist that enable unparalleled measurements of RNA structure, interactions, and performance, and 4) RNA circuits should retain function across diverse organisms because they rely on universal RNA:RNA base pairing interactions.

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 strand, enabling the output to participate in downstream TMSD reactions. TMSD cascades can be programmed via sequence complementarity to execute logic, signal amplification, and complex information processing and decision making. The Cellular Engineering Group has recently developed cotranscriptionally encoded RNA strand displacement (ctRSD) circuits, a technology that enables the genetic encoding of RNA TMSD components. We have demonstrated that ctRSD circuits have predictable and tunable dynamics in vitro and have programmed these circuits to execute a wide range of computational tasks. To facilitate construction of larger ctRSD circuits, we have compiled a toolkit of functional sequences and design principles and developed open-source software for simulating circuit dynamics and generating sequences to test experimentally. Ultimately, ctRSD circuits could be genetically encoded for continuous operation in living cells, cell lysates, or biological samples. In these environments, ctRSD circuits could sense changing patterns of endogenous nucleic acid sequences in real-time and regulate gene expression in response.

In support of RNA circuit engineering, the Cellular Engineering Group is also developing techniques to measure RNA structure, RNA interactions, and RNA circuit dynamics in diverse environments.

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

An animation 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

1. Schaffter, S.W., Wintenberg, M.E., Murphy, T.M., Strychalski, E.A. Design approaches to expand the toolkit for building cotranscriptionally encoded RNA strand displacement circuits. bioRxiv. 2023. DOI: https://doi.org/10.1101/2023.02.01.526534
2. 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
3. Schaffter, S. W.; Murphy, T. M. ctRSD_simulator_2.0. https://ctrsd-simulator.readthedocs.io/en/latest/

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Sam Schaffter Interviewed on Meet the Molecular Programmer Podcast: https://podcast.molpi.gs/media/schaffter-s-1c8799780ad4af6f/