Modified TdT Enzymes for Template-Independent DNA Synthesis
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Solution Overview
Problem
Current DNA synthesis methods are limited in producing strands longer than 200 nucleotides due to inefficiencies in nucleotide coupling, requiring laborious and costly processes like the 'synthesize and stitch' technique, and lack the ability to use 3′-O-reversibly terminated nucleotides for de novo single-stranded DNA synthesis.
Innovation Solution
Modified terminal deoxynucleotidyl transferase (TdT) enzymes with specific amino acid modifications, such as at positions T160, E174, C179, M183, and others, that enhance the incorporation of 3′-O-reversibly terminated nucleotides for improved nucleic acid synthesis, allowing for longer DNA strands and more efficient de novo synthesis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Length of moving object
If synthetic chemistry (phosphoramidite technology) is used to couple nucleotides one at a time, then DNA strands can be synthesised, but the length is limited to 120-200 nucleotides due to mathematical impossibility of achieving acceptable yields beyond this length
Solution Approach 1:
The patent introduces a terminal deoxynucleotidyl transferase (TdT) enzyme as an intermediary biological catalyst to replace the chemical phosphoramidite coupling system. This enzyme-mediated approach allows for template-independent DNA synthesis with significantly improved yields at lengths exceeding 200 nucleotides, overcoming the mathematical yield limitations of stepwise chemical coupling
Solution Approach 2:
The patent modifies the synthesis mechanism by changing from chemical coupling parameters to enzymatic reaction parameters. The TdT enzyme operates under different kinetic and thermodynamic conditions, enabling efficient incorporation of nucleotides at positions beyond 200 where chemical coupling efficiency drops below acceptable thresholds
2Length of moving object
If 'synthesize and stitch' technique is used to produce longer DNA strands, then strands of up to 3 kb can be produced, but the process becomes laborious and costly requiring multiple overlapping fragments
Solution Approach 1:
The patent extracts and eliminates the complex 'synthesize and stitch' multi-step process by using TdT enzyme to directly synthesise long DNA strands in a single continuous reaction. This removes the need for separate fragment synthesis, purification, and assembly steps that characterize the conventional approach
Solution Approach 2:
The TdT enzyme performs preliminary action by directly extending DNA strands from short primers to full-length products in one continuous enzymatic reaction, rather than requiring preliminary synthesis of multiple overlapping fragments that must later be assembled
3Ease of operation
If template-dependent DNA polymerases are used for sequencing-by-synthesis, then strands of 500-1000 bps can be produced, but de novo single-stranded DNA synthesis cannot be achieved
Solution Approach 1:
The patent inverts the conventional template-dependent approach by using template-independent TdT enzyme to synthesise DNA de novo. Instead of requiring a template strand to guide synthesis, the enzyme directly builds single-stranded DNA from nucleotide substrates, enabling applications where template availability is limited
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
These modified enzymes significantly improve the yield and length of synthesized DNA strands, enabling the production of longer sequences and reducing the need for template-dependent methods, thus overcoming the limitations of existing DNA synthesis technologies.
Implementation Method 1
Modified terminal deoxynucleotidyl transferase (TdT) enzymes with specific amino acid modifications, such as at positions T160, E174, C179, M183, and others, that enhance the incorporation of 3′-O-reversibly terminated nucleotides for improved nucleic acid synthesis
Data Source
AI summary
The invention relates to the use of specific terminal deoxynucleotidyl transferase (TdT) enzymes or the homologous amino acid sequence of PoIµ, PoIβ, PoIλ, and PoIθ of any species or the homologous amino acid sequence of X family polymerases of any species in a method of nucleic acid synthesis, to methods of synthesizing nucleic acids, and to the use of kits comprising said enzymes in a method of nucleic acid synthesis. The invention also relates to the use of terminal deoxynucleotidyl transferases or homologous enzymes and 3′-blocked nucleoside triphosphates in a method of template independent nucleic acid synthesis.