Modified Oligonucleotide Assembly Using Ligase-Joined Short Fragments
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing methods for producing oligonucleotides, such as siRNA, face challenges with low purity and yield as the length of the oligonucleotide strand increases, and there is a lack of efficient methods for enzymatic condensation of short oligonucleotide fragments with modified nucleotides.
Innovation Solution
A method involving the enzymatic condensation of four or more oligonucleotide raw material fragments using an oligonucleotide ligase to produce modified oligonucleotides with a complementary portion, achieving higher production efficiency and purity, particularly for strands shorter than 28 nucleotides.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If chemical synthesis method (solid phase synthesis) is used to produce oligonucleotides, then the oligonucleotides can be produced, but purity and yield decrease as the length of the oligonucleotide strand becomes longer
Solution Approach 1:
The patent divides the target oligonucleotide into multiple short fragments (e.g., 4-8 nucleotides each) that can be independently synthesized with high purity using chemical synthesis methods. These fragments are then enzymatically condensed to form the full-length oligonucleotide, avoiding the accumulation of errors that occurs in traditional long-chain chemical synthesis.
Solution Approach 2:
The patent introduces enzymatic condensation (using ligases such as T4 DNA ligase or T4 RNA ligase) as an intermediary step to join the synthesized fragments. This enzymatic process serves as a mediator that accurately assembles the fragments into the target sequence with high fidelity, overcoming the limitations of direct chemical synthesis for long strands.
2Ease of manufacture
If chemical synthesis method is used to produce oligonucleotides, then the oligonucleotides can be produced, but production efficiency is low
Solution Approach 1:
The patent segments the synthesis process into two parallel stages: (1) independent chemical synthesis of multiple short fragments, and (2) enzymatic condensation of these fragments. This segmentation allows for optimized production of each fragment type and parallel processing, significantly improving overall production efficiency compared to sequential chemical synthesis of the entire strand.
Solution Approach 2:
The patent performs preliminary chemical synthesis of multiple short oligonucleotide fragments before the final assembly step. These pre-synthesized fragments are prepared in advance with appropriate overhangs or cohesive ends, enabling efficient enzymatic condensation in a subsequent single step, thereby improving production efficiency.
3Adaptability or versatility
If oligonucleotide raw material comprising short and modified type nucleotide is used for enzymatic condensation, then modified oligonucleotides can be produced, but annealing property is low
Solution Approach 1:
The patent applies modified nucleotides locally at specific positions within the oligonucleotide fragments rather than uniformly throughout the entire sequence. This localized modification strategy maintains the annealing properties of the unmodified regions while introducing desired functional modifications at specific sites, thus balancing versatility and reliability.
Solution Approach 2:
The patent divides the modified oligonucleotide into multiple short fragments, allowing modified nucleotides to be incorporated in a controlled manner within each fragment. The short length of each fragment ensures adequate annealing capability, while the modifications provide the desired functionality. The fragments are then assembled enzymatically to form the complete modified oligonucleotide.
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 enables efficient production of modified oligonucleotides like siRNA and heteroduplex oligonucleotides with high purity and improved yield, overcoming the limitations of traditional chemical synthesis.
Implementation Method 1
enzymatically condensing the annealed oligonucleotide raw material fragments with each other
Implementation Method 2
linking an oligo-DNA fragment and a PEGylated oligo-DNA fragment with a DNA ligase at the cohesive ends thereof
Implementation Method 3
annealing a plurality of oligonucleotide raw material fragments corresponding to fragments of a target oligonucleotide with a template oligonucleotide that is complementary to the target oligonucleotide
Implementation Method 4
complementary sequence
Data Source
Figure 1
Figure 2-1
Figure 2-2
AI summary
The present invention provides a method for efficiently producing an oligonucleotide comprising a complementary portion, such as an siRNA and a heteroduplex oligonucleotide. More specifically, provided is a method for producing a modified oligonucleotide comprising a complementary portion having 11 to 27 nucleotide length, in which the method includes formation of the modified oligonucleotide by treating four or more oligonucleotide raw material fragments in total in the presence of an oligonucleotide ligase; the four or more oligonucleotide raw material fragments in total correspond to oligonucleotide raw material fragments that are obtained by dividing the modified oligonucleotide at a fragment linking site that satisfies following conditions (i) to (v): (i) one or more fragment linking sites are present in the complementary portion in each strand side, and two or more fragment linking sites in total are present in the modified oligonucleotide; (ii) when the modified oligonucleotide is divided at the fragment linking site, a sticky end is formed in the complementary portion, in which the sticky end has 1 to 10 nucleotide length; (iii) at least one oligonucleotide raw material fragment has a modified nucleotide; (iv) four oligonucleotide raw material fragments out of the four or more oligonucleotide raw material fragments in total include the complementary portion having 5 to 25 nucleotide length; and (v) total nucleotide length of the oligonucleotide raw material fragments corresponding to the complementary portions in each strand side is 11 to 27.