Cotranscriptionally encoded RNA strand displacement circuits

Abstract

Engineered molecular circuits that process information in biological systems could address emerging human health and biomanufacturing needs. However, such circuits can be difficult to rationally design and scale. DNA-based strand displacement reactions have demonstrated the largest and most computationally powerful molecular circuits to date but are limited in biological systems due to the difficulty in genetically encoding components. Here, we develop scalable cotranscriptionally encoded RNA strand displacement (ctRSD) circuits that are rationally programmed via base pairing interactions. ctRSD circuits address the limitations of DNA-based strand displacement circuits by isothermally producing circuit components via transcription. We demonstrate circuit programmability in vitro by implementing logic and amplification elements, as well as multilayer cascades. Furthermore, we show that circuit kinetics are accurately predicted by a simple model of coupled transcription and strand displacement, enabling model-driven design. We envision ctRSD circuits will enable the rational design of powerful molecular circuits that operate in biological systems, including living cells.

DNA strand displacement based computational systems and their applications

“…With the help of transcriptase, DSD system can be deployed in cells. Schaffter et al (Schaffter and Strychalski, 2022) designed a DNA strand with a specific sequence that can be transcript to RNA complexes in cells. This specific DNA strand is endocytosed by cells, and then transcripted in cells.…”

“…DSD can be combined with other technologies and utilized in a wide range of applications. So far, DSD has been combined with CRISPR technology (Ishino et al, 1987;Montagud-Martínez et al, 2021), DNA origami (Rothemund, 2006;Zhang et al, 2022), enzymes (Bucci et al, 2022;Schaffter and Strychalski, 2022), proteins , etc. These combinations effectively expand the application scenarios for DSD.…”

Design approaches to expand the toolkit for building cotranscriptionally encoded RNA strand displacement circuits

“…To investigate the influence of input branch migration length on gate performance, we tested gates with several extended input branch migration lengths. Previously, 16 base branch migration lengths were used 20 but this relatively short duplex could dehybridize in environments with low salt concentrations or elevated temperatures. Here, we tested G{u1,w2r} with (18, 20, and 22) base input branch migration lengths and found all gates had the expected performance.…”

“…Strand displacement exposes a toehold domain on the outputs that can initiate downstream strand displacement reactions with other ctRSD gates or reporters (Figure 1A). Based on these principles, ctRSD circuits have been programmed to execute logic operations and multilayer signaling cascades with predictable kinetics in in vitro transcription reactions 20 . Because all ctRSD circuit components are produced together via transcription, these circuits could be genetically encoded for continuous operation in living cells, cell lysates, or biological samples, where nucleases would eventually deplete TMSD components added at fixed concentrations 13,21,22 .…”

“…Cotranscriptionally encoded RNA strand displacement (ctRSD) circuits 20 are an emerging technology, modeled after state-of-the-art, DNA-based TMSD circuits 18,19 , with the potential for implementation across many synthetic biology platforms. In ctRSD circuits, DNA templates are transcribed to produce single-stranded RNA (ssRNA) inputs and double-stranded RNA (dsRNA) gates.…”