Nucleotide Strand Synthesis Complex With Nick-Sealing Ligase
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Solution Overview
Problem
Existing nucleotide strand synthesis methods result in non-target strands that cannot be effectively separated, particularly for lengths greater than 20 mer, affecting the function and efficacy of nucleotide strands, especially in antisense strand drugs, RNAi drugs, and nucleic acid aptamer drugs.
Innovation Solution
A complex using nucleotide fragments with specific phosphate and hydroxyl groups synthesized in alternating directions, forming double-stranded oligonucleotides with a ligase to seal nicks, reducing non-target strands and improving purity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If chemical synthesis is used to synthesize nucleotide strands, then the synthesis process is simple and fast, but the purity of the synthesized product decreases as the length of the nucleotide strand increases
Solution Approach 1:
The nucleotide strand synthesis is divided into two independent synthesis processes: synthesizing the first nucleotide strand and synthesizing the second nucleotide strand. Each strand is synthesized separately with controlled length, then the two strands are annealed to form the final double-stranded product. This segmentation allows each synthesis step to achieve high purity while maintaining overall productivity.
Solution Approach 2:
The patent combines two separately synthesized nucleotide strands through annealing to form the final double-stranded nucleotide product. By merging two high-purity single strands, the final product achieves high purity without requiring the entire long strand to be synthesized in one step, thus resolving the contradiction between synthesis speed and purity.
2Manufacturing precision
If purification processes are applied to remove non-target strands, then the purity of the synthesized product is improved, but the complexity of the synthesis process increases
Solution Approach 1:
The patent performs preliminary actions by synthesizing two separate nucleotide strands with controlled lengths and purities before the final annealing step. By preparing high-purity single strands in advance, the final assembly step produces high-purity double-stranded products without requiring complex purification processes, thus improving purity while avoiding increased process complexity.
3Productivity
If non-target nucleotide strands are present in the synthesized product, then the synthesis efficiency is maintained, but the function and activity of the nucleotide strands of interest are affected
Solution Approach 1:
The synthesis process is segmented into two independent strands, each synthesized with controlled length and high purity. This segmentation ensures that only the desired nucleotide sequences are produced, eliminating non-target strands that would compromise functionality, while maintaining synthesis efficiency through parallel processing of both strands.
Solution Approach 2:
The patent changes the synthesis parameters by controlling the length and purity of each individual nucleotide strand during synthesis. By optimizing parameters such as synthesis length and purity for each strand, the final annealed product achieves high reliability and functionality without sacrificing synthesis efficiency.
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
The method significantly reduces non-target nucleotide strands, enhancing the purity and efficacy of synthesized nucleotide strands by forming intact phosphodiester bonds.
Implementation Method 1
the nucleotide fragments are hybridized to form a double-stranded oligonucleotide
Implementation Method 2
forming intact phosphodiester bonds
Implementation Method 3
the double-stranded oligonucleotide ligase is a ligase capable of sealing nicks in the double-stranded oligonucleotide
Implementation Method 4
sealing nicks in the double-stranded oligonucleotide
Data Source
AI summary
The present disclosure provides a complex for synthesizing one or more nucleotide strands of interest and a method for synthesizing one or more nucleotide strands of interest. The complex for synthesizing one or more nucleotide strands of interest includes nucleotide fragments and a double-stranded oligonucleotide ligase, wherein the nucleotide fragments are hybridized to form a double-stranded oligonucleotide; the double-stranded oligonucleotide ligase is a ligase capable of sealing nicks in the double-stranded oligonucleotide; and at least one strand of the double-stranded oligonucleotide is a nucleotide strand of interest; and the nucleotide fragments include a first fragment and a second fragment, the first fragment contains a 5′-phosphate group, and the first fragment is synthesized in a 3′- to 5′-direction; and the second fragment contains a 3′-hydroxyl group, and the second fragment is synthesized in a 5′- to 3′-direction.