Single-Stranded Nucleic Acid Production via Host Cell Replication
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Solution Overview
Problem
Current methods for producing single-stranded nucleic acids, such as ssDNA, face challenges in large-scale production with high sequence integrity and low error rates, particularly for longer sequences, which is crucial for pharmaceutical applications like cancer treatment where high-quality aptamers are needed.
Innovation Solution
A method involving the replication and amplification of a repetitive cluster of a desired target nucleic acid sequence within a vector, followed by cloning into host cells for replication and subsequent degradation to produce high-quality single-stranded nucleic acids with extended sequences, utilizing restriction sites and polymerase chain reaction for accuracy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If phosphoramidite chemistry is used for ssDNA production, then large-scale production is achieved, but synthesis errors increase with sequence length
Solution Approach 1:
The patent segments the ssDNA production process into two distinct phases: (1) in vivo synthesis of double-stranded DNA using cellular replication machinery, and (2) in vitro conversion to single-stranded DNA using exonuclease treatment. This segmentation allows each phase to leverage its respective strengths - cellular systems for high-fidelity replication and enzymatic methods for clean single-strand conversion - thereby achieving both large-scale production and high sequence accuracy.
Solution Approach 2:
The patent introduces double-stranded DNA as an intermediary form between the desired single-stranded product and the starting materials. By first producing dsDNA through in vivo replication (which ensures high fidelity) and then converting to ssDNA through controlled exonuclease treatment, the method uses dsDNA as a mediator to bridge the gap between scalable production and high sequence accuracy requirements.
2Productivity
If phosphoramidite chemistry is used for ssDNA production, then large-scale production is achieved, but error rate increases
Solution Approach 1:
The patent employs the host cell's own replication machinery to synthesize the DNA sequence of interest. The cellular polymerase systems naturally possess high fidelity and proofreading capabilities, allowing the system to self-correct errors during replication. This self-service approach leverages the inherent reliability of biological systems to produce high-integrity sequences at scale, eliminating the need for error-prone chemical synthesis for each copy.
Solution Approach 2:
Double-stranded DNA serves as an intermediary that protects sequence integrity during amplification. The dsDNA form allows for high-fidelity in vivo replication, and the subsequent enzymatic conversion to ssDNA preserves this fidelity while enabling the desired single-stranded product format.
3Reliability
If aptamer sequences are extended to over 100 nucleotides, then binding specificity is improved, but synthesis errors increase
Solution Approach 1:
The patent segments the synthesis approach by using in vivo replication for the full-length sequence rather than chemical synthesis. This allows extended sequences (>100 nt) to be produced as continuous, high-fidelity copies through cellular machinery, avoiding the cumulative errors that occur with stepwise chemical addition of nucleotides.
Solution Approach 2:
The patent changes the fundamental parameter of synthesis mechanism from chemical (phosphoramidite) to biological (in vivo replication). This parameter change enables the production of extended sequences with high accuracy, as cellular polymerases maintain low error rates even over long replication distances, unlike chemical synthesis where errors accumulate with each coupling step.
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 method enables the production of high-quality single-stranded nucleic acids with extended sequences, achieving high sequence accuracy and large-scale production that conventional methods cannot achieve, suitable for sequences over 100 nucleotides, enhancing the reliability of aptamers for pharmaceutical applications.
Implementation Method 1
replication and amplification of a repetitive cluster of a desired target nucleic acid sequence within a vector, followed by cloning into host cells for replication
Implementation Method 2
utilizing restriction sites and polymerase chain reaction for accuracy
Data Source
AI summary
The invention relates to a method for production of single-stranded macronucleotides by amplifying and ligating an extended monomeric single-stranded target nucleic acid sequence (targetss) into a repetitive cluster of double-stranded target nucleic acid sequences (targetds), and subsequently cloning the construct into a vector (aptagene vector). The aptagene vector is transformed into host cells for replication of the aptagene and isolated in order to optain single-stranded target sequences (targetss). The invention also relates to single-stranded nucleic acids, produced by a method of the invention.