Engineered TdT Variants for Blocked-Nucleotide DNA Synthesis
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Solution Overview
Problem
Current methods for synthesizing long polynucleotides are inefficient and generate toxic waste, and existing template-independent enzymatic synthesis techniques using terminal deoxynucleotidyl transferase (TdT) are hindered by blocked 3′-OH groups that prevent further nucleotide addition and large cleavable linkers that interfere with synthesis.
Innovation Solution
Engineered TdT polypeptides with specific amino acid substitutions and improved thermostability, allowing template-independent synthesis of polynucleotides using nucleoside triphosphates with 3′-O-removable blocking groups, enabling defined sequence synthesis without a complementary template.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of substance
If traditional phosphoramidite DNA synthesis is used, then short oligonucleotides can be produced, but the method generates high amounts of toxic waste and is limited to approximately 200 basepairs
Solution Approach 1:
The patent replaces chemical phosphoramidite synthesis methods with enzymatic synthesis using engineered TdT polypeptides. This substitution eliminates the need for toxic phosphoramidite reagents and harsh chemical conditions, thereby reducing toxic waste generation while enabling efficient synthesis of long polynucleotides through biological catalysis
2Manufacturing precision
If 3′-OH blocked NTPs are used in template-independent synthesis, then stepwise addition of specific nucleotide residues is enabled, but the blocked 3′-OH prevents further nucleotide addition
Solution Approach 1:
The patent uses 3′-OH blocked NTPs where the blocking group is attached in advance to the 3′-OH position. This preliminary blocking enables controlled stepwise incorporation of nucleotides with defined sequences, while the blocking group can be removed after each incorporation to restore the 3′-OH for the next addition cycle
Solution Approach 2:
The synthesis process employs periodic cycles of nucleotide incorporation followed by blocking group removal. Each cycle consists of adding a blocked NTP, allowing incorporation, then removing the blocking group to enable the next addition, creating a rhythmic pattern of controlled synthesis steps
3Adaptability or versatility
If large cleavable linkers are used to attach blocking groups, then removable blocking functionality is achieved, but the linkers interfere with synthesis
Solution Approach 1:
The patent modifies the parameters of the blocking group attachment by using small, minimally intrusive linkers instead of large cleavable linkers. This parameter change maintains the removability function while reducing steric interference with the enzyme active site and improving synthesis accuracy by preventing unwanted interactions during the polymerization process
4Productivity
If wild-type TdT is used for template-independent synthesis, then nucleoside addition occurs, but the activity is limited by blocked 3′-OH groups
Solution Approach 1:
The patent changes the biochemical parameters of TdT by engineering amino acid substitutions that modify the enzyme's interaction with blocked NTP substrates. These parameter changes increase catalytic activity toward blocked substrates while maintaining fidelity and reliability of the synthesis process through improved enzyme-substrate compatibility
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 TdT polypeptides facilitate the efficient production of long polynucleotides with reduced by-product formation and increased activity, overcoming the limitations of traditional synthesis methods.
Implementation Method 1
engineered terminal deoxynucleotidyl transferase (TdT) polypeptides useful in template-independent polynucleotide synthesis
Data Source
AI summary
The present invention provides engineered terminal deoxynucleotidyl transferase (TdT) polypeptides useful in template-independent polynucleotide synthesis using a nucleoside triphosphate-3′-O-removable blocking group (NTP-3′-O-RBG), as well as compositions, methods of utilizing these engineered polypeptides, and polynucleotides encoding the engineered terminal deoxynucleotidyl transferases.