Polymerase Stabilization via Zwitterionic and Cationic Ester Compounds
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Solution Overview
Problem
Stabilization of polymerases for nucleic acid synthesis and amplification is challenging for long-term storage, especially at room temperature, as existing methods require lyophilization and specific detergents, and the enzymes are prone to inactivation at temperatures above freezing.
Innovation Solution
Development of liquid compositions containing polymerases and non-detergent zwitterionic or cationic ester compounds that stabilize the enzymes at room temperature for extended periods without the need for lyophilization or cellulose matrices, maintaining activity comparable to storage at lower temperatures.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If polymerases are stored at room temperature, then storage convenience is improved, but polymerase stability deteriorates due to inactivation
Solution Approach 1:
The patent introduces non-detergent zwitterionic compounds (such as phosphocholine and phosphoserine) and cationic ester compounds as intermediary substances that mediate between the polymerase enzyme and the storage environment. These compounds form protective complexes with the polymerase, preventing thermal inactivation at room temperature while maintaining enzymatic activity, thus resolving the contradiction between storage convenience and enzyme stability
Solution Approach 2:
The patent changes the chemical parameters of the storage medium by incorporating specific zwitterionic and cationic ester compounds at optimized concentrations. This parameter change transforms the storage conditions from detrimental (room temperature without stabilizers) to beneficial (room temperature with protective compounds), allowing convenient storage while maintaining polymerase stability and activity
2Reliability
If lyophilization is used to stabilize polymerases, then storage stability is improved, but device complexity increases due to additional processing steps
Solution Approach 1:
The patent extracts and eliminates the need for lyophilization processing by introducing chemical stabilizers that provide sufficient protection at room temperature. This removes the complex freeze-drying step from the workflow, reducing device complexity while maintaining storage stability through the action of zwitterionic and cationic ester compounds
Solution Approach 2:
The patent replaces the expensive and time-consuming lyophilization process with simpler, chemically-based stabilization using relatively inexpensive zwitterionic and cationic ester compounds. This substitution achieves comparable or superior stability without requiring complex equipment or energy-intensive processing
3Reliability
If detergents are used to stabilize polymerases, then storage stability is improved, but harmful factors increase due to detergent toxicity
Solution Approach 1:
The patent converts the potentially harmful detergent stabilization approach into a beneficial non-detergent zwitterionic compound system. These compounds provide similar stabilizing effects through different mechanisms that do not involve the toxic properties of traditional detergents, thus eliminating harmful factors while maintaining storage stability
Solution Approach 2:
The patent changes the chemical nature of the stabilizing agents from ionic detergents to zwitterionic and cationic ester compounds. This parameter change in chemical composition eliminates the harmful toxic effects associated with detergents while preserving the stabilizing function, allowing safe room temperature storage without compromising polymerase activity
Data Source
AI summary
Described herein are stabilized polymerase compositions comprising a polymerase and an polymerase stabilizing agent, such as a non-detergent zwitterionic stabilizer or a cationic ester disclosed, for use in nucleic acid amplification or nucleic acid sequencing. Compositions are provided for the stabilization of one or more polymerases in a single stabilized liquid formulation. Also disclosed are methods for making and using stabilized polymerase compositions and kits for nucleic acid amplification and sequencing comprising the stabilized polymerase compositions provided.