Modified Oligonucleotide Assembly Using Ligated Short Fragments
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Solution Overview
Problem
Existing methods for producing oligonucleotides, such as siRNA, face challenges with low purity and yield, especially for longer strands, and there is a lack of efficient enzymatic condensation methods for short oligonucleotide fragments with modified nucleotides.
Innovation Solution
A method involving the use of four or more oligonucleotide raw material fragments and an oligonucleotide ligase to enzymatically construct double-strand oligonucleotides with lengths less than 28 nucleotides, enhancing purity and production efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If a solid phase synthesis method is used to produce oligonucleotides, then the oligonucleotides can be produced by linear extension one nucleotide at a time, but the 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., 4-10 nucleotides each) that can be independently synthesized with high purity. These fragments are then enzymatically condensed using ligase to form the complete oligonucleotide. This segmentation approach avoids the cumulative error and purity loss associated with long-chain solid phase synthesis, as each short fragment can be synthesized with high fidelity and then assembled with precise enzymatic control.
2Ease of manufacture
If a solid phase synthesis method is used to produce oligonucleotides, then the oligonucleotides can be produced by linear extension one nucleotide at a time, but the production efficiency is low
Solution Approach 1:
The patent performs preliminary synthesis of multiple short oligonucleotide fragments in parallel, each independently produced with high efficiency. These pre-synthesized fragments are then ready for enzymatic condensation in a subsequent step. This preliminary action allows the system to bypass the time-consuming sequential extension of long chains, as the fragments are already prepared and can be rapidly assembled through enzymatic ligation, significantly improving overall production efficiency.
3Productivity
If short oligonucleotide fragments with modified nucleotides are used for enzymatic condensation, then the annealing property is reduced, but the target oligonucleotide needs to be produced
Solution Approach 1:
The patent modifies physical and chemical parameters of the enzymatic condensation process to accommodate fragments with modified nucleotides. This includes optimizing reaction temperature, pH, buffer composition, and enzyme concentration to enhance the annealing and ligation efficiency of modified fragments. By adjusting these parameters, the system maintains high production efficiency while ensuring reliable annealing properties despite the presence of modified nucleotides that would otherwise reduce stability.
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 allows for the efficient production of modified oligonucleotides like siRNA and heteroduplex oligonucleotides with high purity and improved yield.
Implementation Method 1
treating four or more oligonucleotide raw material fragments in the presence of an oligonucleotide ligase to thereby construct the double strand oligonucleotide
Data Source
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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.