Design Approaches to Expand the Toolkit for Building Cotranscriptionally Encoded RNA Strand Displacement Circuits

Area of Science:

• Molecular biology
• Synthetic biology
• Biochemistry

Background:

• Cotranscriptionally encoded RNA strand displacement (ctRSD) circuits offer programmable molecular computation.
• Current ctRSD circuits are limited by a small number of characterized components, restricting circuit size and capabilities.
• Applications range from in vitro diagnostics to cellular computation.

Purpose of the Study:

• To expand the toolkit of ctRSD components by characterizing over 200 gate sequences.
• To identify and mitigate failure modes in ctRSD gate design.
• To enable the development of larger and more complex ctRSD circuits.

Main Methods:

• Systematic characterization of over 200 ctRSD gate sequences.
• Exploration of variations in input, output, and toehold sequences.
• Investigation of design parameters including domain lengths, ribozyme sequences, and transcription order.
• Identification of failure modes and development of design strategies to reduce failures.

Main Results:

• A library of over 200 ctRSD gate sequences was characterized, expanding the available components.
• Enabled circuits with up to four times more inputs compared to previous designs.
• Identified specific failure modes and developed systematic design approaches to enhance robustness.
• Demonstrated that ctRSD gate design is robust to variations in transcriptional encoding.

Conclusions:

• The expanded toolkit and design approaches significantly enhance the capabilities and potential applications of ctRSD circuits.
• ctRSD circuits can be engineered for increased complexity and reliability.
• This work broadens the design space for ctRSD applications in diverse and complex environments.