Engineered RNA Ligases for Scalable Modified Oligonucleotide Synthesis
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Solution Overview
Problem
Chemical synthesis of oligonucleotides for therapeutics faces challenges such as low efficiency, difficulty in scale-up, and environmental impact, while existing RNA ligases have limitations in ligating polynucleotides with modified sugar residues and non-standard internucleoside linkages.
Innovation Solution
Engineered RNA ligase polypeptides with specific amino acid sequences and substitutions, designed to enhance efficiency and promiscuity in ligating polynucleotides with modified sugar residues and non-standard internucleoside linkages, offering improved activity and stability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If chemical synthesis is used to produce oligonucleotides, then oligonucleotides can be manufactured, but the synthesis efficiency is low and scaling up is difficult
Solution Approach 1:
The patent replaces chemical synthesis methods with an enzymatic system using engineered RNA ligase. The ligase enzyme catalyzes the ligation of oligonucleotide fragments to form full-length oligonucleotides, substituting chemical reactions with a biological catalytic system that offers better scalability and efficiency.
Solution Approach 2:
The patent introduces amino acid substitutions at specific positions (e.g., positions 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100) in the RNA ligase sequence to enhance its catalytic activity and substrate binding affinity, thereby improving synthesis efficiency and enabling scale-up.
2Productivity
If traditional RNA ligase is used to ligate polynucleotides with modified sugar residues and non-standard internucleoside linkages, then ligation can occur, but the efficiency and substrate tolerance are limited
Solution Approach 1:
The patent employs multiple amino acid substitutions in the RNA ligase sequence to modify the enzyme's active site geometry and chemical properties. These changes enable the enzyme to accommodate and efficiently process diverse substrates including those with modified sugar residues (e.g., 2′-O-methyl, 2′-O-ethyl, 2′-fluoro) and non-standard internucleoside linkages (e.g., phosphorothioate), significantly expanding substrate tolerance while maintaining high ligation efficiency.
Solution Approach 2:
The engineered RNA ligase is designed to perform multiple ligation functions across a broad range of substrates. The enzyme can ligate polynucleotides with various sugar modifications, different internucleoside linkages, and diverse sequence compositions, making it a universal tool for synthesizing modified oligonucleotides that traditional ligases cannot efficiently process.
3Productivity
If chemical synthesis with toxic solvents is used, then oligonucleotides can be produced, but toxic chemical waste is generated
Solution Approach 1:
The patent replaces toxic chemical synthesis processes with an aqueous enzymatic ligation system. The engineered RNA ligase catalyzes oligonucleotide assembly in water-based buffers, eliminating the need for organic solvents such as toluene and acetonitrile, thereby preventing toxic chemical waste generation while maintaining manufacturing capability.
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 engineered RNA ligases demonstrate increased efficiency and stability, enabling effective synthesis of modified oligonucleotides with enhanced substrate tolerance and yield, addressing the limitations of chemical synthesis and traditional RNA ligases.
Implementation Method 1
engineered RNA ligase polypeptides... for the synthesis of polynucleotides... methods of using the recombinant RNA ligase... for nucleic acid synthesis
Data Source
AI summary
The present disclosure relates to engineered RNA ligase polypeptides and compositions thereof, as well as polynucleotides encoding the engineered RNA ligase polypeptides. The present disclosure also provides methods of using the engineered RNA ligase polypeptides or compositions thereof for molecular biological, diagnostic, and other purposes.