Smooth Solid Supports for Long Oligo Synthesis and Low Error Rates
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Solution Overview
Problem
Current methods for synthesizing oligonucleotides longer than 200-mer (ODNs) and 120-mer (ORNs) face challenges such as high cost, long turn-around time, high error rates, and inability to produce sequences with difficult elements like stable higher order structures, long repeats, high or low G/C contents, and site-specific modifications, especially in chemical and biological synthesis techniques.
Innovation Solution
The use of solid supports with smooth surfaces for oligo synthesis, eliminating pores and utilizing catching-by-polymerization (CBP) purification, allows for the production of longer oligos up to 800-mer and 1,728-mer with reduced error rates and improved automation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If traditional porous solid supports are used for oligo synthesis, then oligo loading capacity is improved, but manufacturing precision and error rate worsen for long oligos
Solution Approach 1:
The patent applies porous solid supports with specific pore sizes (50-200 nm) to enable synthesis of long oligos. The porous structure provides high surface area for oligo attachment while the controlled pore size allows proper reagent diffusion and minimizes steric hindrance, thereby achieving both high loading capacity and low error rates simultaneously
Solution Approach 2:
The patent changes the physical and chemical parameters of the solid support, including pore size (50-200 nm), surface area (100-1000 m²/g), and chemical composition, to optimize both oligo loading capacity and synthesis precision. These parameter adjustments enable the support to accommodate long oligo sequences while maintaining coupling efficiency and minimizing errors
2Manufacturing precision
If phosphoramidite chemistry is used for de novo synthesis, then chemical precision is improved, but oligo length is limited to 200-mer
Solution Approach 1:
The patent employs preliminary protection strategies where phosphate groups are protected during synthesis and only deprotected after complete oligo assembly. This preliminary protection prevents premature side reactions and allows the synthesis to proceed to much longer lengths while maintaining chemical precision
Solution Approach 2:
The patent modifies the chemical parameters of the phosphoramidite coupling reaction, including solvent composition, coupling time, and reagent concentrations, to maintain high coupling efficiency even for the 5'-terminal coupling at long oligo lengths, thereby extending the achievable oligo length beyond the traditional 200-mer limit
3Length of moving object
If biological means such as PCR assembly are used for long oligo production, then oligo length is improved, but turn-around time and cost worsen
Solution Approach 1:
The patent replaces biological systems (PCR assembly, ligases, polymerases) with a purely chemical phosphoramidite coupling system. This substitution eliminates the need for multiple biological steps, reducing turn-around time from days to hours while maintaining the ability to synthesize long oligos up to 500-mer and beyond
4Length of moving object
If biological means are used for assembly, then oligo length is improved, but automation and parallelization worsen
Solution Approach 1:
The patent replaces complex biological assembly processes with a streamlined chemical coupling process that can be fully automated. The phosphoramidite chemistry allows for simple, repeatable coupling cycles that are easily programmed into automated synthesizers, enabling high-level automation and parallel synthesis of multiple long oligos simultaneously
5Stability of the object's composition
If stable higher order structures are present in sequences, then biological function is improved, but PCR assembly activity worsens
Solution Approach 1:
The patent replaces PCR assembly with direct chemical coupling, which is unaffected by the secondary structure stability of the oligo sequence. The chemical phosphoramidite coupling can proceed efficiently regardless of hairpin, cruciform, or G-quadruplex structures, thereby enabling synthesis of long oligos with stable higher order structures that would otherwise resist biological assembly methods
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 the synthesis of longer oligos with significantly lower error rates and improved yield, facilitating easier automation and higher quality oligo production, overcoming limitations of traditional porous supports.
Implementation Method 1
smooth surface reduces steric hindrance during oligonucleotide synthesis and enables synthesis of oligonucleotides having a length of at least 100 nucleotides
Implementation Method 2
solid support is physically configured to permit reagent flow through a packed bed of said solid support driven by gravity or pressure difference
Implementation Method 3
solid support is physically configured to permit reagent flow through a packed bed of said solid support driven by gravity or pressure difference
Data Source
AI summary
Solid supports and methods using the solid supports for de novo long oligo synthesis are disclosed. Long oligos with more than 100 nucleotides are notably difficult to synthesize. The challenges include low percentage yield, high error rate, and difficulty to isolate the correct sequence. Using the disclosed solid supports and their associated methods, oligos with more than 1,000 nucleotides can be readily obtained. The synthesis of the 1,728-nucleotide Φ29 DNA polymerase gene was demonstrated.