RNA Ligase Assembly of Non-Natural Oligonucleotides at High Purity
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for synthesizing oligonucleotides, particularly non-natural RNA strands, are inefficient, costly, and difficult to scale up, with low yields and high impurity levels, especially as strand length increases.
Innovation Solution
A method using RNA ligases from families Rnl1, Rnl2, Rnl3, and Rnl5 to ligate non-natural ribonucleotides through phosphodiester bonds, forming double-stranded nucleic acid structures with nicks, which are then ligated to create continuous strands.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If solid-phase synthesis method is used to synthesize oligonucleotides, then the oligonucleotides can be produced, but the yield decreases with the increase of strand length and the purification process becomes cumbersome
Solution Approach 1:
The patent divides the long RNA strand into multiple shorter substrates (e.g., 20-30 nt each) that are synthesized separately with high purity, then ligates them together using RNA ligase. This segmentation allows each substrate to be produced with high purity through solid-phase synthesis, while the final long strand assembly maintains overall purity without requiring extensive purification of the entire long strand during synthesis.
Solution Approach 2:
The patent uses RNA ligase as an intermediary enzyme to join the pre-synthesized RNA substrates together. The ligase mediates the phosphodiester bond formation between substrates, enabling high-purity assembly without the need for complex purification steps that would be required if the entire long strand were synthesized in one continuous process.
2Length of moving object
If the strand length is increased during solid-phase synthesis, then longer oligonucleotides are obtained, but the yield and purity decrease significantly
Solution Approach 1:
The patent segments the long strand into multiple shorter substrates of manageable length (20-30 nt each), which can be synthesized with high yield through solid-phase synthesis. By ligating these high-yield substrates together, the overall long strand is obtained with much higher total yield than attempting to synthesize the entire long strand in one continuous solid-phase synthesis reaction.
Solution Approach 2:
The patent performs preliminary synthesis of multiple short RNA substrates separately before assembling them into the final long strand. This preliminary action allows each substrate to be optimized for high-yield synthesis independently, and then they are combined through ligation, resulting in superior overall yield compared to direct synthesis of the full-length strand.
3Length of moving object
If the strand length is increased during solid-phase synthesis, then longer oligonucleotides are obtained, but the purification process becomes cumbersome and cost increases
Solution Approach 1:
The patent segments the synthesis process into substrate production and assembly phases. The substrates are synthesized with high purity, and the ligation step uses the inherent complementarity of the substrates to ensure correct assembly. This approach simplifies purification compared to synthesizing long strands directly, as the substrates can be purified once at a manageable scale, and the ligation step requires minimal additional purification.
4Productivity
If RNA ligase is used to ligate non-natural RNA strands, then synthesis efficiency can be improved, but existing RNA ligases cannot efficiently link non-natural RNA strands
Solution Approach 1:
The patent modifies parameters of the RNA ligase including amino acid sequence mutations and structural optimizations to enable efficient ligation of non-natural RNA substrates. These parameter changes allow the ligase to accommodate and efficiently process non-natural ribonucleotides while maintaining catalytic activity, thereby achieving both high synthesis efficiency and reliable ligation of non-natural strands.
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 approach enhances synthesis efficiency, reduces costs, and allows for large-scale production of non-natural RNA strands with high purity, overcoming the limitations of traditional solid-phase synthesis.
Implementation Method 1
the nicks are ligated by a phosphodiester bond with the RNA ligase
Implementation Method 2
an RNA substrate is ligated with an RNA ligase, to obtain an oligonucleotide
Data Source
AI summary
Provided is a method for preparing an oligonucleotide with a ribonucleic acid (RNA) ligase. The RNA ligase includes any one or more enzymes of RNA ligase families Rnll, Rn12, Rn13, and Rn15, and the oligonucleotide includes natural RNA or non-natural RNA. The method may solve the problem that in the prior art, it is difficult to efficiently synthesize a non-natural RNA strand, and the method is suitable for the field of RNA synthesis.