Near-Threshold Translational Repressors for High-Fold Gene Silencing
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Solution Overview
Problem
Current synthetic biology lacks systems that effectively demonstrate a decrease in gene expression upon triggering, limiting the ability to achieve strong repression of target gene expression.
Innovation Solution
Development of synthetic nucleic acid molecules with near-threshold hairpin structures and trigger recognition sequences that allow for a translationally active or inactive state in response to a repressing trigger RNA, enabling a high fold-change in protein expression regulation through the use of near-threshold translational repressors (NeaTTRs) and logic circuits like NAND and NOR gates.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional riboregulators are used to decrease gene expression upon triggering, then repression of target gene expression can be achieved, but the repression strength is insufficient and fold-change is limited
Solution Approach 1:
The patent applies parameter changes by designing hairpin structures with specific thermodynamic parameters (near-threshold stability) that allow the RNA to exist in equilibrium between folded and unfolded states. By carefully adjusting the hairpin stability parameters (Delta G values near threshold), the system achieves high fold-change repression responses to trigger RNA binding, resolving the contradiction between reliable repression and high productivity.
Solution Approach 2:
The invention implements dynamics by creating a dynamic RNA switch that can transition between two stable states (folded hairpin with repressed translation and unfolded state with active translation). The near-threshold design allows the system to dynamically respond to trigger RNA concentration changes, achieving both strong repression reliability and high fold-change through the dynamic equilibrium between states.
2Adaptability or versatility
If de novo design for riboregulators is used to increase gene expression in response to trigger RNA, then activation capability is improved, but systems demonstrating inverse behavior (decrease in gene expression) have yet to be reported
Solution Approach 1:
The patent applies inversion by designing a system where trigger RNA binding induces hairpin folding that represses translation, rather than the conventional approach where trigger binding activates expression. This inverted mechanism places the repressed state as the default (folded hairpin sequestering RBS) and uses trigger binding to maintain or enhance repression, achieving reliable inverse behavior that was previously unreported.
3Measurement precision
If near-threshold hairpin structures are designed to enable high fold-change repression, then expression control precision is improved, but system complexity increases due to multiple trigger recognition sequences
Solution Approach 1:
The patent applies merging by combining multiple functional elements into a single integrated near-threshold hairpin structure. The hairpin simultaneously incorporates the ribosomal binding site, start codon, and multiple trigger recognition sequences (first and second recognition sequences) within its structure. This merging achieves precise expression control through the coordinated action of all elements while avoiding the complexity of separate modular components.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The solution achieves significant repression of gene expression with high fold-change ratios and orthogonality, enabling precise control of protein levels and integration into complex logic circuits for applications in diagnostics and metabolic engineering.
Implementation Method 1
the hairpin structure is configured to alternatively adopt a translationally active ON state or adopt a translationally inactive OFF state in the presence of a repressing trigger RNA, and wherein the second trigger recognition sequence does not encode an in-frame stop codon
Data Source
AI summary
Provided herein are synthetic nucleic acid molecules and methods of using such synthetic nucleic acid molecules for strong repression of target gene expression. In particular, provided herein are methods for altering expression of a protein in a cell, where the method comprises introducing into a cell a protein coding sequence operably linked to a near-threshold translational repressor having first and second trigger recognition sequences that are fully or partially complementary to a repressing trigger RNA; and introducing into a cell the repressing trigger RNA.