Fusion Protein Gene Expression Control via Small Molecule Dimerization
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Solution Overview
Problem
Current post-translational control systems for gene expression have high background expression levels, which can be undesirable in various applications, and there is a need for improved systems with reduced background expression, enhanced packaging, transduction, promoter design, and vector design.
Innovation Solution
The development of fusion proteins comprising a DNA binding domain operably linked to a dimerization domain, utilizing specific sequences from domains like Gal4, zinc-finger proteins, Cas9, and Cas12a, to regulate gene expression by binding to response elements, with the ability to modulate transcriptional or epigenetic activities through small molecule-induced dimerization.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If post-translational control systems use small molecule-induced dimerization to regulate gene expression, then temporal modulation and control are achieved, but background expression levels are high
Solution Approach 1:
The system divides the transcription factor into two separate fusion proteins: one containing the DNA binding domain and the other containing the transcriptional activation domain. Each fusion protein has a dimerization domain that can bind to the small molecule. Only when both fusion proteins are present and bound to the small molecule do they form a functional transcription factor complex, thereby reducing background expression while maintaining temporal modulation control.
Solution Approach 2:
The small molecule acts as an intermediary that mediates the dimerization between the two fusion proteins. The small molecule binds to dimerization domains on both fusion proteins, bringing them together to form the active transcription factor complex only when the small molecule is present, thus controlling gene expression temporally while minimizing background activity.
2Ease of operation
If fusion proteins use dimerization domains for small molecule binding, then gene expression control is achieved, but system complexity increases
Solution Approach 1:
The dimerization domains are designed to be universal binding units that can recognize and bind to the same small molecule class. This allows different fusion proteins to be combined in various configurations while maintaining a consistent control mechanism, reducing the overall system complexity despite the modular nature of the 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
This approach reduces background expression, allows for precise control of gene expression, and improves the overall efficiency and specificity of gene regulation, enabling effective applications in gene therapy and cell signaling.
Implementation Method 1
Chemically induced dimerization (CID) is one mechanism by which a small molecule can be used to effect post translational control of expression of the gene of interest. These systems make use of a small molecule to induce dimerization of proteins and thereby localize components required for transcription.
Implementation Method 2
The fusion protein comprises a DNA binding domain that specifically binds to a response element, wherein the DNA binding domain comprises a sequence derived from one or more of a galactose-activated transcription factor 4 (Gal4) sequence, a zinc-finger 1 (ZF1) sequence, a zinc-finger 2 (ZF2) sequence, a zinc-finger 3 (ZF3) sequence, a zinc finger HIV2 (ZFHIV2) sequence, a zinc-finger homeodomain 1 (ZFHD1) sequence, a catalytically inactive Cas12a (dCas12a) sequence, a catalytically inactive Cas9 (dCas9) sequence, and a catalytically inactive CasPhi (dCasPhi) sequence.
Data Source
AI summary
A fusion protein comprising a DNA binding domain operably-linked to a dimerization domain, wherein the DNA binding domain specifically binds to a response element.