Nucleotide Fragment Ligation for High-Purity Long Oligonucleotide Synthesis
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Solution Overview
Problem
Existing nucleotide strand synthesis methods result in non-target strands that are difficult to separate, particularly for lengths greater than 20 mer, affecting the purity 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 terminal groups (5'-phosphate and 3'-hydroxyl) and a double-stranded oligonucleotide ligase to form double-stranded oligonucleotides, sealing nicks and reducing non-target strands.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If chemical synthesis is used to synthesize nucleotide strands, then the synthesis process is simple and straightforward, 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 segments: synthesis of the nucleotide strand of interest and synthesis of the complementary strand. Each segment is synthesized separately with high purity, then the two segments are ligated together. This segmentation allows each synthesis step to achieve high purity independently, resolving the contradiction between synthesis simplicity and product purity for long strands.
Solution Approach 2:
The two independently synthesized segments (nucleotide strand of interest and complementary strand) are merged through ligation to form the complete double-stranded nucleotide strand. This merging approach combines the advantages of simple chemical synthesis for each segment with the high purity achieved through separate synthesis, ultimately producing high-purity long nucleotide strands.
2Manufacturing precision
If purification processes are applied to remove non-target strands, then the purity of the synthesized product can be improved, but the separation effectiveness decreases for non-target strands with lengths close to the target strand
Solution Approach 1:
The complementary strand is synthesized separately as an independent component, then ligated to the nucleotide strand of interest. This extraction approach separates the synthesis of the target strand from the synthesis of the complementary strand, allowing each to be optimized for high purity without relying on difficult separations of closely sized non-target strands.
Solution Approach 2:
The complementary strand is synthesized in advance as a separate component with high purity, before ligation to the nucleotide strand of interest. This preliminary action ensures that both components entering the ligation step are of high purity, preventing the formation of non-target strands with lengths close to the target, thereby eliminating the need for difficult purification separations.
3Ease of manufacture
If non-target nucleotide strands are present in the synthesized product, then the synthesis process remains straightforward, but the efficacy of the nucleotide strands is seriously affected
Solution Approach 1:
The synthesis process is segmented into independent synthesis of the nucleotide strand of interest and its complementary strand, followed by ligation. This segmentation ensures that each segment is synthesized with high purity, preventing the formation of non-target strands that would compromise efficacy, while maintaining relative simplicity through standard chemical synthesis methods.
Solution Approach 2:
The high-purity nucleotide strand of interest and high-purity complementary strand are merged through ligation to form the final high-purity double-stranded product. This merging of two high-purity components ensures the final product has high purity and maintains efficacy, while the ligation step remains a simple and straightforward process.
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
Significantly improves the purity of nucleotide strands by reducing non-target strands, enhancing the efficacy of nucleotide strands, particularly in longer sequences.
Implementation Method 1
the double-stranded oligonucleotide ligase is a ligase capable of sealing nicks in the double-stranded oligonucleotide
Implementation Method 2
the nucleotide fragments are hybridized to form a double-stranded oligonucleotide
Data Source
AI summary
A composition for synthesizing a nucleotide chain and a synthesis method for a nucleotide chain, which belong to the technical field of nucleotides. The composition for synthesizing a nucleotide chain comprises a nucleotide fragment and a double-stranded ligase. The nucleotide fragment is used for forming a double-stranded structure; the double-stranded ligase is a ligase capable of sealing a nick in the double-stranded structure; and at least one strand of the double-stranded structure is a target nucleotide chain. The nucleotide fragment comprises a first fragment and a second fragment; the 5' end of the first fragment contains a monophosphate group, and the first fragment is synthesized in the 3' to 5' direction; and the 3' end of the second fragment contains a hydroxyl group, and the second fragment is synthesized in the 5' to 3' direction.