Double-Stranded Concatemeric DNA for Cell-Free Expression
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing in vitro transcription-translation systems require large quantities of DNA templates, which are labor-intensive and costly to produce, especially for eukaryotic cell-free protein expression, and are inefficient due to the need for high concentrations of template DNA in larger reaction volumes.
Innovation Solution
The use of double-stranded concatemeric DNA generated through rolling circle amplification (RCA) with a minimalistic expression sequence, including a promoter, cap-independent translation element (CITE), and open reading frame (ORF), which can be used at lower concentrations (0.1 ng/µL to 35 ng/µL) in eukaryotic cell-free expression systems, reducing the need for PCR-based synthesis and minimizing unwanted sequence expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If traditional PCR-based DNA synthesis is used to generate template DNA for IVTT, then sufficient quantities of DNA can be obtained, but the process is labor-intensive, time-consuming, and requires multiple workflow steps
Solution Approach 1:
The invention extracts only the essential expression sequence from the full plasmid DNA, eliminating unnecessary cloning steps, origin of replication sequences, and antibiotic resistance genes. This extraction of the core functional element (expression sequence) enables direct use as template DNA without time-consuming PCR amplification or cloning procedures.
Solution Approach 2:
The expression sequence is pre-prepared and purified before being introduced into the IVTT reaction. This preliminary preparation of the minimalistic expression sequence eliminates the need for in-situ DNA synthesis during the protein expression workflow, saving significant time and reducing procedural complexity.
2Quantity of substance
If traditional plasmid cloning and propagation in E. coli is used, then high-scale DNA production is achieved, but the process is time-consuming and labor-intensive
Solution Approach 1:
The invention removes the plasmid backbone elements (origin of replication, antibiotic resistance markers, cloning sites) and retains only the minimalistic expression sequence. This extraction eliminates the need for E. coli propagation and cloning steps, enabling rapid DNA production without traditional bacterial culture workflows.
Solution Approach 2:
The minimalistic expression sequence serves as a disposable, single-use template that does not require maintenance through bacterial propagation. Unlike plasmids that need continuous culture expansion, the purified expression sequence can be directly used in IVTT reactions without further biological amplification steps.
3Productivity
If dialysis is used to remove waste products in mammalian cell-free extract, then protein expression efficiency is enhanced, but the reaction volume increases significantly and template DNA becomes unrecoverable
Solution Approach 1:
The invention changes the DNA template parameters by using a minimalistic expression sequence with optimized composition and structure. This modified template design achieves efficient protein expression in mammalian cell-free extracts without requiring dialysis-mediated waste removal, thereby avoiding volume expansion and DNA loss.
4Loss of time
If linear PCR products are used as DNA templates, then DNA can be generated quickly, but the linear DNA is rapidly degraded by nucleases in cell-free extracts
Solution Approach 1:
The invention extracts the expression sequence and presents it in a format (purified DNA fragment or minimalistic plasmid) that is inherently more resistant to nuclease degradation compared to linear PCR products. The extracted sequence can be used with minimized nuclease activity in the IVTT system, ensuring template stability throughout the reaction.
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 enables efficient and cost-effective cell-free protein expression with lower DNA template requirements, improving yield and reducing the complexity of DNA template production, while maintaining high protein expression levels even at lower DNA concentrations.
Implementation Method 1
Isothermal DNA amplification techniques such as rolling circle amplification (RCA) can be employed to generate large quantities of high-quality DNA with less effort, time, and expense.
Implementation Method 2
a desired protein of interest is expressed by adding a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) that encodes a gene of the protein of interest to a transcription-translation-competent cellular extract, and performing the transcription and/or translation of the gene of interest
Implementation Method 3
performing the transcription and/or translation of the gene of interest
Data Source
Figure 1
Figure 2
Figure 3
AI summary
Methods for in vitro transcription and translation using a double-stranded concatemeric DNA in a eukaryotic cell-free expression system are provided. The method includes the steps of (a) contacting a double-stranded concatemeric DNA with a eukaryotic cell-free expression system, and (b) expressing a protein in vitro from the double-stranded concatemeric DNA in the eukaryotic cell-free expression system. The double-stranded concatemeric DNA includes a plurality of tandem repeat sequences.The plurality of tandem repeat sequences includes an expression sequence including a promoter, a cap-independent translation element (CITE), and an open reading frame. A final concentration of the double-stranded concatemeric DNA in the eukaryotic cell-free expression system is in a range from about 0.1 ng/µLto about 35 ng/µL. A RCA product DNA may be used as the double stranded concatemer DNA for the methods.