Modified TdT Variants for Template-Free Nucleotide Synthesis
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Solution Overview
Problem
Existing methods for nucleic acid synthesis, such as solid-phase phosphoramidite chemistry and template-independent DNA polymerases, face limitations in incorporating modified nucleotides efficiently, leading to poor synthesis quality, high costs, and limited fragment lengths.
Innovation Solution
Development of modified Terminal deoxynucleotidyl Transferase (TdT) variants with targeted amino acid substitutions, allowing for the incorporation of modified nucleotides into nucleic acid fragments without a template, enhancing synthesis efficiency and reducing costs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If native TdT is used for template-independent synthesis, then the enzyme can synthesize nucleic acids without template, but it is poorly able to use modified nucleotides
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid substitutions at positions 173, 302, and 313 in the TdT enzyme sequence. These substitutions modify the enzyme's biochemical parameters to enable efficient incorporation of modified nucleotides while maintaining template-independent synthesis capability, directly resolving the contradiction between versatility and reliability
Solution Approach 2:
The patent applies local quality by making targeted modifications at specific locations (residues 173, 302, 313) within the TdT protein structure. These localized changes in specific regions allow the enzyme to recognize and incorporate modified nucleotides without affecting the overall template-independent synthesis function, thereby improving adaptability while preserving reliability
2Length of moving object
If solid-phase phosphoramidite chemistry is used for nucleic acid synthesis, then multiple solvents and reagents are required, but the length of synthetic oligonucleotides is limited to 150-200 bases
Solution Approach 1:
The patent replaces the chemical synthesis mechanism (phosphoramidite chemistry requiring multiple solvents and reagents) with an enzymatic mechanism using modified TdT. This substitution eliminates the need for complex chemical reagent systems while enabling synthesis of longer oligonucleotides beyond the 150-200 base limit, resolving both the length limitation and device complexity issues
3Ease of operation
If template-independent DNA polymerases are used to add modified nucleotides, then one type of nucleotide can be added per cycle in controlled fashion, but the incorporation efficiency is insufficient
Solution Approach 1:
The patent applies parameter changes by modifying specific amino acid residues (173, 302, 313) in the polymerase enzyme to enhance its catalytic efficiency for incorporating modified nucleotides. These changes improve the enzyme's ability to recognize and process modified nucleotides while maintaining controlled addition, thereby resolving the contradiction between ease of operation and productivity
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 modified TdT variants enable faster and more cost-effective synthesis of nucleic acids with predetermined sequences by efficiently incorporating modified nucleotides, improving synthesis quality and reducing turnaround time.
Implementation Method 1
The invention relates to variants of Terminal deoxynucleotidyl Transferase (TdT) and uses thereof for the enzymatic synthesis of nucleic acid sequences without template
Data Source
AI summary
The present invention relates to variants of Terminal deoxynucleotidyl Transferase (TdT), each of which (i) has an amino acid sequence similarity to SEQ ID NO: 2, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 or 35 with corresponding amino acid substitutions, (ii) is capable of synthesizing a nucleic acid fragment without a template and (iii) is capable of incorporating a modified nucleotide into the nucleic acid fragment.