N-Terminally Truncated TdT Variants for Solid Support Synthesis
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Solution Overview
Problem
Current template-free enzymatically-based polynucleotide synthesis methods using terminal deoxynucleotidyl transferase (TdT) enzymes are inefficient in incorporating reversibly blocked nucleoside triphosphates and struggle with accessing the interior of solid supports due to the enzyme's size and stability limitations.
Innovation Solution
Development of N-terminally truncated TdT variants with stabilizing mutations and interior affinity tags that reduce the enzyme's radius of gyration, allowing for enhanced access to solid supports and improved efficiency in incorporating reversibly blocked nucleoside triphosphates.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If wild type TdT is used for template-free polynucleotide synthesis, then the enzyme maintains natural stability and structure, but it cannot efficiently incorporate reversibly blocked nucleoside triphosphates and cannot access the interior of solid supports
Solution Approach 1:
The TdT enzyme is divided into two parts: an N-terminal truncation that reduces the radius of gyration for better solid support penetration, and a C-terminal catalytic domain that retains enzymatic function. This segmentation allows the enzyme to access interior spaces of solid supports while maintaining catalytic activity.
Solution Approach 2:
The physical parameters of the TdT enzyme are changed by removing the N-terminal region, which reduces the radius of gyration from approximately 40-50 Å in wild type to approximately 20-30 Å in the truncated variant. This parameter change enables the enzyme to penetrate into the interior of solid supports.
2Length of moving object
If the TdT enzyme is truncated to reduce its size for solid support access, then it can penetrate interior spaces more easily, but it loses structural stability
Solution Approach 1:
Stability-enhancing mutations are introduced specifically at the N-terminus of the truncated enzyme, where the structural impact is minimal. The mutation Q152E (in mouse TdT numbering) locally enhances stability through improved packing and electrostatic interactions without disrupting the overall folded structure or catalytic function.
Solution Approach 2:
The stabilizing mutation is introduced in advance to compensate for the destabilizing effect of N-terminal truncation. This preemptive stabilization ensures that the truncated enzyme maintains sufficient structural integrity and thermal stability for practical use in solid-phase synthesis.
3Reliability
If natural TdT is used, then it maintains natural catalytic function, but it incorporates reversibly blocked nucleoside triphosphates with greatly reduced efficiency
Solution Approach 1:
The catalytic parameters of TdT are optimized through the N-terminal truncation, which alters the enzyme's conformational dynamics and substrate binding properties. This structural modification enhances the catalytic efficiency for incorporating reversibly blocked nucleoside triphosphates while retaining the ability to synthesize polynucleotides without a template.
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 TdT variants demonstrate increased efficiency in synthesizing polynucleotides on solid supports by reducing steric interference and improving stability, enabling more effective template-free enzymatic nucleic acid synthesis.
Implementation Method 1
template-free polymerases, such as terminal deoxynucleotidyl transferase (TdT), because of the proven efficiency of such enzymes
Implementation Method 2
a stabilizing amino acid is a substitution for the glutamine at position 4 (or a functionally equivalent position) which increases the melting temperature of a TdT variant by at least 1° C.
Implementation Method 3
compact TdT variants having a minimized size and radius of gyration which permits the TdT variants to access the interior spaces of solid supports and resins more readily
Data Source
AI summary
The present invention is directed to terminal deoxynucleotidyl transferase (TdT) variants from a variety of species which have been engineered for increase compactness and enhanced efficiency in incorporating reversibly blocked nucleoside triphosphates into a polynucleotide, especially polynucleotides attached porous solid supports. The invention includes use of such TdTs for synthesizing polynucleotides of any predetermined sequence.