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DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation
Published on: December 29, 2021
Ligand-Induced Trigger RNA Cleavage Enables Programmable Gene Expression Regulation via Strand Displacement in
Chenxi Zhang1, Huiye Feng1, Liting Li1
1MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry, Sun Yat-Sen University, 510275 Guangzhou, Guangdong, China.
Background:
Synthetic RNA circuits offer powerful tools for reprogramming cellular behavior, but constructing ligand-responsive RNA switches that function reliably inside living cells remains challenging. Existing cis-acting designs often lack modularity and programmability due to tight coupling between sensing and output domains.
Methods:
We developed a generalizable strategy termed Ligand-Induced Trigger RNA Cleavage (LITC). This approach integrates an aptamer-embedded hammerhead ribozyme (aptazyme) as a trans-acting trigger. The aptazyme sequence is inserted into the spacer region of an RNA trigger strand, separating its toehold and displacement domains. Ligand-induced aptazyme self-cleavage inactivates the trigger, thereby controlling downstream toehold-mediated strand displacement reactions. We validated this system in both prokaryotic (E. coli) and eukaryotic (HEK-293T) cells using translation-controlling toehold switches and gRNA switches within the CRISPR/Cas9 system.
Results:
The LITC strategy successfully enabled programmable, dose-dependent regulation of gene expression. A spacer inserted between toehold and displacement domains did not impair trigger function. Embedding self-cleaving ribozymes (HHR, sTRSV) constitutively silenced trigger activity. Using a theophylline-responsive aptazyme, we achieved ligand-controlled regulation of a toehold switch, with different communication modules (CMs) yielding varied regulatory performance and theophylline concentrations up to 4 mM providing graded control. Furthermore, this approach was extended to control CRISPR interference (CRISPRi) in E. coli and CRISPR activation (CRISPRa) of the endogenous ASCL1 gene in HEK-293T cells, demonstrating cross-system portability.
Conclusions:
The LITC platform provides a general, modular, and transferable strategy for small-molecule control of toehold-mediated strand displacement reactions. It enables precise conditional regulation of RNA-based devices, including translation switches and CRISPR-Cas9 systems, across both prokaryotic and eukaryotic cells, thereby offering a versatile framework for constructing intelligent genetic circuits.
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