Double-Stranded Concatemeric DNA for Stable Protein Expression
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Solution Overview
Problem
Current RNA and protein expression systems using eukaryotic cell lines and rolling circle amplification (RCA) products face low expression rates and high costs, necessitating a more efficient method for producing high-quality RNA and protein with improved yields and reduced production costs.
Innovation Solution
A method involving the use of double-stranded concatemeric DNA with phosphorothioated nucleotides, generated through rolling circle amplification, is introduced for RNA or protein expression in eukaryotic cells, where the DNA includes a plurality of tandem repeat sequences with a specific ratio of phosphorothioated nucleotides to total nucleotides, enhancing stability and expression efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional plasmid DNA is used for in vivo protein production, then stable cell lines can be generated, but the process requires large volumes of cell culture and long production time
Solution Approach 1:
The patent segments the DNA into concatemeric structures with multiple copies of the gene of interest arranged in tandem repeats, allowing high-level expression without requiring stable integration into the genome, thus reducing production time while maintaining reliability
Solution Approach 2:
The patent performs preliminary action by generating double-stranded concatemeric DNA with phosphorothioated nucleotides before transfection, which enhances DNA stability and expression efficiency, allowing shorter culture periods to achieve desired protein yields
2Quantity of substance
If traditional plasmid DNA is used for large scale production, then sufficient DNA template is available, but manufacturing becomes expensive and cumbersome with contamination risks
Solution Approach 1:
The patent extracts only the essential expression sequence from the plasmid, eliminating unnecessary bacterial maintenance elements, and uses rolling circle amplification to generate concatemeric DNA directly, simplifying manufacturing while maintaining sufficient DNA quantity
Solution Approach 2:
The patent changes the chemical structure of nucleotides by incorporating phosphorothioated nucleotides at specific ratios (1:1600 to 125:1600), which enhances DNA stability and reduces contamination susceptibility without complicating the manufacturing process
3Ease of manufacture
If RCA product DNA is used for RNA and protein expression, then production cost is reduced, but expression rate is low resulting in low yields
Solution Approach 1:
The patent creates a composite DNA structure by combining rolling circle amplification products with phosphorothioated nucleotides, maintaining the cost advantages of RCA while enhancing expression rate through the stabilizing and immunomodulatory effects of phosphorothioate modifications
Solution Approach 2:
The patent optimizes the ratio of phosphorothioated nucleotides to total nucleotides (1:1600 to 125:1600) to achieve the desired balance between cost-effectiveness and high expression rate, with higher ratios providing enhanced stability and expression
4Adaptability or versatility
If plasmid DNA is used for transient production, then lethal proteins can be expressed, but large quantities of RNA or protein require larger scale cell culture
Solution Approach 1:
The patent uses composite concatemeric DNA structures with enhanced stability from phosphorothioated nucleotides, achieving high-level transient expression that reduces the cell culture volume needed to produce large quantities of protein while maintaining the ability to express lethal proteins
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 approach results in higher and more stable expression of proteins, such as GFP and RFP, with improved cell culture stability and yield compared to traditional plasmid DNA methods, reducing production costs and increasing the viability of RNA and protein products.
Implementation Method 1
The double-stranded concatemeric DNA comprises one or more phosphorothioated nucleotides, wherein a ratio of phosphorothioated nucleotides to total nucleotides in the double-stranded concatemeric DNA is in a range of 1:1600 to 125:1600
Implementation Method 2
Isothermal DNA amplification techniques such as rolling circle amplification (RCA) may be employed to generate such large quantities of high-quality DNA with less effort, time, and expense, starting from a circular nucleic acid template
Data Source
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AI summary
A method for in vivo RNA or protein expression is provided. The method includes introducing a double-stranded concatemeric DNA into a eukaryotic cell to generate a desired RNA or protein. The double-stranded concatemeric DNA includes a plurality of tandem repeat sequences, wherein each of the plurality of tandem repeat sequences comprises an expression sequence. The double-stranded concatemeric DNA comprises one or more phosphorothioated nucleotides, wherein a ratio of phosphorothioated nucleotides to total nucleotides in the double-stranded concatemeric DNA is at least 1:1600. A eukaryotic cell comprising an exogeneous, double-stranded concatemeric DNA comprising the plurality of tandem repeat sequences is also provided.