Segmented Enzymatic Assembly for Scalable Oligonucleotide Production
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Solution Overview
Problem
Existing methods for producing oligonucleotides and polynucleotides, particularly at large scales, face challenges such as scale-up limitations, high costs, and increased error rates due to sequential addition of nucleotides and complex purification processes, which are inefficient and time-consuming.
Innovation Solution
A process involving the use of polymerases and ligases to anneal segment polynucleotides to a complementary template, extend gaps with nucleoside triphosphates, and ligate them to form single- or double-stranded products, reducing the need for sequential nucleotide addition and complex purification steps.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If solid-supported synthesis is used to produce oligonucleotides, then the method is well-established and can be automated, but scale-up limitations occur and batch sizes are restricted
Solution Approach 1:
The invention divides the oligonucleotide synthesis into two main stages: (1) synthesis of multiple short oligonucleotide segments using solid-supported chemistry, and (2) ligation of these segments to form the final full-length oligonucleotide. This segmentation allows the use of established solid-phase synthesis for creating segments while overcoming its scale-up limitations through enzymatic ligation, thereby achieving both manufacturing reliability and scalability.
Solution Approach 2:
The invention combines solid-supported synthesis with enzymatic ligation in a hybrid approach. The solid-supported synthesis step creates the oligonucleotide segments using well-established chemistry, while the enzymatic ligation step merges these segments into the final product. This merging allows the benefits of both methods to be realized: the reliability of solid-phase synthesis and the scalability of enzymatic processes.
2Manufacturing precision
If sequential addition of nucleotides is used in solid-phase synthesis, then defined sequences can be produced, but errors accumulate with increasing length
Solution Approach 1:
The invention segments the long oligonucleotide into multiple shorter segments, each synthesized using solid-supported chemistry with high precision. By keeping each segment relatively short, the error accumulation during sequential nucleotide addition is minimized. The segments are then ligated together to form the final long oligonucleotide, thereby maintaining high sequence accuracy even for long products.
Solution Approach 2:
The invention uses enzymatic ligation as an intermediary step to join the synthesized segments. This enzymatic process serves as a mediator that connects the individually synthesized segments with high fidelity, reducing the overall error rate compared to direct sequential synthesis of the entire long oligonucleotide in one process.
3Productivity
If multiple rounds of synthesis are performed to overcome scale-up limitations, then larger batch sizes can be achieved, but the process becomes more time-consuming
Solution Approach 1:
The invention segments the synthesis process into parallelizable steps: multiple short oligonucleotide segments can be synthesized simultaneously in parallel using solid-supported chemistry, and then ligated together in a single enzymatic step. This segmentation enables concurrent production of multiple segments, significantly reducing the total time required compared to sequential synthesis rounds.
Solution Approach 2:
The invention maintains continuous useful action by using enzymatic ligation to join segments in a single step after synthesis. The enzymatic process continuously processes all segments simultaneously, eliminating the need for multiple sequential rounds of synthesis and purification, thereby reducing overall production time while maintaining large batch sizes.
4Manufacturing precision
If chromatography is used for purification of crude oligonucleotide, then purity can be achieved, but the process becomes expensive and lengthy at large scales
Solution Approach 1:
The invention extracts and removes the complex chromatography purification step from the process by using enzymatic ligation. The enzymatic process inherently provides purification through selective ligation of correct segments, eliminating the need for separate chromatography steps. This extraction of the purification function into the ligation step itself reduces both complexity and cost at large scales.
Solution Approach 2:
The enzymatic ligation process provides self-service purification by selectively joining only the correct oligonucleotide segments while leaving incorrect ones unreacted. This self-selective process eliminates the need for external chromatography purification systems, reducing device complexity and operational costs while maintaining high product purity.
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 enhances production efficiency by minimizing errors and reducing complexity, allowing for cost-effective and scalable synthesis of modified oligonucleotides and polynucleotides, suitable for therapeutic applications.
Implementation Method 1
contacting a template polynucleotide, which comprises a sequence complementary to the single-stranded polynucleotide product, with a pool of at least two segment polynucleotides under conditions to allow annealing of the at least two segment polynucleotides to the template polynucleotide
Implementation Method 2
extending at least one of the annealed segment polynucleotides using a pool of nucleoside triphosphates and a polymerase, to fill in the at least one sequence gap
Implementation Method 3
ligating segment polynucleotide(s) and/or extended segment polynucleotide(s) using a ligase to form the single-stranded polynucleotide product bound to the template polynucleotide in a duplex
Implementation Method 4
changing the conditions to denature the duplex comprising the single-stranded polynucleotide product and the template polynucleotide, whereby the single-stranded polynucleotide product is produced
Data Source
AI summary
The invention relates to novel processes using enzymes for the production of polynucleotides or oligonucleotides, wherein said processes are suitable for use in the production of modified polynucleotides or oligonucleotides, such as those for use in therapy.