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

Published

May 3, 2023

Author(s)

Samuel Schaffter, Molly Wintenberg, Terence Murphy, Elizabeth Strychalski

Abstract

Cotranscriptionally encoded RNA strand displacement (ctRSD) circuits are an emerging tool for programmable molecular computation, with potential applications spanning in vitro diagnostics to continuous computation inside living cells. In ctRSD circuits, RNA strand displacement gates and inputs are continuously produced together via transcription. These RNA components can be rationally programmed through base pairing interactions to execute logic and signaling cascades. However, the small number of ctRSD components characterized to date limits circuit size and capabilities. Here, we characterize 220 ctRSD gate sequences, exploring different input, output, and toehold sequences and changes to other design parameters, including domain lengths, ribozyme sequences, and the order in which gate strands are transcribed. This characterization provides a library of sequence domains for engineering ctRSD components, i.e., a toolkit, potentially enabling circuits with up to four-fold more inputs than previously possible. We also identify specific failure modes and systematically develop design approaches that reduce the likelihood of failure across different gate sequences. Lastly, we show ctRSD gate design is robust to changes in transcriptional encoding, opening a broad design space for applications in more complex environments. Together, these results deliver an expanded toolkit and design approaches for building ctRSD circuits that will dramatically extend capabilities and potential applications.

Citation

ACS Synthetic Biology

Pub Type

Journals

Download Paper

https://doi.org/10.1021/acssynbio.3c00079

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Citation

Schaffter, S. , Wintenberg, M. , Murphy, T. and Strychalski, E. (2023), Design approaches to expand the toolkit for building cotranscriptionally encoded RNA strand displacement circuits, ACS Synthetic Biology, [online], https://doi.org/10.1021/acssynbio.3c00079, https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=935989 (Accessed April 25, 2024)