Mutant Pol Theta Polymerase for Template-Free Nucleic Acid Extension
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing DNA polymerases, particularly those of the Pol theta subfamily, are limited in their ability to incorporate a diverse range of nucleic acid analogs and generate long polymers, which hinders the development of high-affinity and specific nucleic acid molecules like aptamers, as they are constrained by their native activity and primer conformation.
Innovation Solution
Engineering mutant DNA polymerases of the Pol theta subfamily with specific amino acid substitutions at positions 2322, 2328, 2334, 2335, 2384, 2387, and 2391, allowing them to incorporate both natural and modified nucleotides, including ribonucleotides, in a template-free manner, resulting in the synthesis of long polymers.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If native DNA polymerase activity is used, then high fidelity replication is achieved, but the ability to incorporate diverse nucleic acid analogs and generate long polymers is limited
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid substitutions at positions 2322, 2328, 2334, 2335, 2384, 2387, and 2391 in the Pol theta polymerase sequence. These mutations alter the enzyme's biochemical parameters to enable incorporation of non-canonocal nucleotides while maintaining polymerase activity, thus resolving the contradiction between fidelity and versatility.
Solution Approach 2:
The engineered polymerase achieves multi-functionality by being capable of incorporating both canonical deoxyribonucleotides and various modified nucleotides (ribonucleotides, nucleic acid analogs) in a single enzyme system. This universal substrate acceptance allows the enzyme to function as both a replicative polymerase and a tool for generating diverse nucleic acid libraries.
2Adaptability or versatility
If template-dependent replication is used, then accurate copying is achieved, but non-templated nucleic acid extension capability is lost
Solution Approach 1:
The patent introduces dynamic behavior to the polymerase by enabling it to switch between template-dependent and template-independent modes of operation. The engineered polymerase can dynamically adapt its activity based on substrate availability and reaction conditions, performing accurate replication when template is present and non-templated extension when template is absent or when random polymer synthesis is required.
3Productivity
If chemical synthesis of random oligonucleotides is used, then combinatorial libraries can be generated, but the process is time-consuming and costly
Solution Approach 1:
The patent replaces the mechanical/chemical synthesis approach with an enzymatic system. Instead of using chemical synthesis methods that require complex solid-phase synthesis machinery and multiple coupling steps, the engineered polymerase catalyzes the formation of nucleic acid polymers in solution, dramatically simplifying the manufacturing process and increasing productivity.
4Adaptability or versatility
If Pol theta polymerase is used with native activity, then DNA replication occurs, but incorporation of ribonucleotides and modified nucleotides is restricted
Solution Approach 1:
The patent applies local quality by making targeted changes at specific positions (2322, 2328, 2334, 2335, 2384, 2387, 2391) within the enzyme's active site and substrate binding regions. These localized mutations alter the chemical environment at specific locations to accommodate diverse nucleotide substrates without requiring complete redesign of the entire enzyme structure, thus increasing substrate specificity while controlling structural complexity.
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 mutant polymerases efficiently produce long homo- or heteropolymers of ribonucleotides and nucleic acid analogs, enhancing the stability, affinity, and specificity of functional nucleic acids, such as aptamers, and enabling the creation of diverse libraries for molecular recognition strategies.
Implementation Method 1
mutant DNA polymerases of the Pol theta subfamily capable of performing non-templated nucleic acid extension
Implementation Method 2
incorporate both natural and modified nucleotides, including ribonucleotides, in a template-free manner, resulting in the synthesis of long polymers
Data Source
AI summary
The invention relates to mutant DNA polymerases of the Pol theta subfamily capable of performing non-templated nucleic acid extension, or of a functional fragment of such a polymerase, methods of producing these mutant DNA polymerases, kits and methods of using these mutant DNA polymerases.