Selective Oligonucleotide Amplification via Rolling Circle Replication
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Solution Overview
Problem
The existing methods for synthesizing oligonucleotides on a large scale are inefficient and costly, resulting in low quantities of high-quality oligonucleotides, which are insufficient for constructing two- and three-dimensional nanoscale DNA structures, and often contain truncated sequences that interfere with the folding process.
Innovation Solution
The use of rolling circle replication and circle-to-circle amplification methods to selectively amplify specific oligonucleotides from a chip-synthesized oligonucleotide pool, producing high-quality oligonucleotides in larger quantities by consecutive rounds of amplification, which can reach up to a billion-fold amplification, and are suitable for use as staple strands in DNA nanostructures.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If chip-based parallel synthesis is used to produce oligonucleotides, then synthesis capacity is improved, but the quantity of oligonucleotides produced is insufficient for DNA nanostructure assembly
Solution Approach 1:
The patent uses rolling circle replication to copy oligonucleotide sequences from chip-synthesized templates, generating billions of copies of each oligonucleotide sequence. This copying process transforms the limited quantity of chip-synthesized oligonucleotides (1 pmol total) into sufficient quantities (micromolar concentrations) for DNA nanostructure assembly
Solution Approach 2:
The patent performs preliminary chip-based synthesis to create template oligonucleotides with unique sequences, then uses these templates to guide the subsequent rolling circle replication process. This preliminary action ensures that only the desired oligonucleotide sequences are amplified in the subsequent steps
2Quantity of substance
If conventional amplification methods are used, then oligonucleotide quantity is improved, but manufacturing precision deteriorates due to truncated sequences
Solution Approach 1:
The patent extracts only the full-length, high-quality oligonucleotide sequences through rolling circle replication, leaving behind truncated sequences and synthesis byproducts. The method selectively amplifies complete oligonucleotide sequences from the chip-synthesized pool, ensuring high manufacturing precision
Solution Approach 2:
The patent uses disposable chip-synthesized template oligonucleotides that are consumed during the rolling circle replication process. These templates are sacrificed to generate large quantities of high-quality product oligonucleotides, separating the low-cost template function from the high-value product
3Quantity of substance
If large quantities of oligonucleotides are synthesized conventionally, then quantity is improved, but cost increases and quality decreases
Solution Approach 1:
Instead of synthesizing each oligonucleotide individually or in large pools, the patent copies specific sequences from a small library of chip-synthesized templates through rolling circle replication. This copying approach is cost-effective because chip-based synthesis is cheaper than conventional synthesis, and the replication process generates large quantities from minimal templates
Solution Approach 2:
The patent segments the oligonucleotide production process into two distinct stages: (1) chip-based synthesis of a diverse template pool, and (2) selective rolling circle replication of specific templates. This segmentation allows optimization of each stage independently, achieving both cost-effectiveness and high quantity
4Manufacturing precision
If oligonucleotide pools contain truncated sequences, then synthesis completeness is improved, but folding reliability deteriorates
Solution Approach 1:
The patent converts the presence of truncated sequences in chip-synthesized pools from a harmful factor into a beneficial selection process. By using rolling circle replication with specific primers, the method selectively amplifies only the complete, full-length oligonucleotide sequences, while truncated sequences remain unamplified and are effectively removed from the final product
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 allows for the cost-effective production of high-quality oligonucleotides in sufficient quantities for DNA nanostructure assembly, reducing defects and improving folding yields, while avoiding the interference of unwanted oligonucleotides.
Implementation Method 1
The invention provides compositions and methods for selective amplification of oligonucleotides from a larger oligonucleotide pool, as well as the resultant oligonucleotide pools and subpools. Embodiments of the invention are based, at least in part, on a nucleic acid amplification method, referred to as rolling circle replication.
Implementation Method 2
the subpool-specific region (1) comprises a subpool-specific sequence and an endonuclease site, and (2) is flanked at each end by a nicking site
Implementation Method 3
the subpool-specific region comprises a subpool-specific sequence and an endonuclease site
Implementation Method 4
Two- and three-dimensional nanoscale shapes can be constructed from folding a long strand of DNA. This folding is based on the specificity of the interactions between complementary nucleotides.
Data Source
AI summary
Provided herein are nucleic acids and methods for selectively amplifying in parallel tens of thousands of high quality oligonucleotides without common sequences. The resultant oligonucleotides can be used for a variety of purposes and applications including but not limited to DNA nano structure synthesis.