Endonuclease V-Cleavable Aptamer for Temperature-Controlled DNA Polymerase Activation
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Solution Overview
Problem
Existing methods for blocking DNA polymerase activity using oligonucleotide aptamers struggle to achieve complete inhibition at low temperatures while allowing full activation at elevated temperatures, as aptamer structures often compromise between these requirements, leading to inefficient DNA synthesis.
Innovation Solution
Incorporating an endonuclease V-cleavable oligonucleotide aptamer that binds to DNA polymerase at low temperatures and is cleaved by endonuclease V enzymatic activity at elevated temperatures, thereby activating DNA synthesis, using a reaction mixture containing DNA polymerase, endonuclease V-cleavable aptamer, and endonuclease V enzymatic activity, with temperature-dependent activation facilitating both activities.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If an oligonucleotide aptamer is used to block DNA polymerase activity at low temperatures, then DNA synthesis inhibition is improved, but complete activation at elevated temperatures is compromised
Solution Approach 1:
The aptamer is designed to bind to and inhibit DNA polymerase before the reaction begins at low temperatures. This preliminary inhibition prevents spurious primer extension and ensures that DNA synthesis only occurs when the aptamer is inactivated by heating to the elevated reaction temperature, thereby resolving the contradiction between initial inhibition and subsequent activation
Solution Approach 2:
The system exploits temperature as a critical parameter to control aptamer-DNA polymerase binding. At low temperatures, the aptamer binds effectively to inhibit polymerase activity. When heated to elevated temperatures, the binding is disrupted, releasing the inhibition and enabling DNA synthesis. This parameter-based control resolves the contradiction by using temperature transitions to switch between inhibition and activation states
2Productivity
If the reaction temperature is increased to activate DNA polymerase, then DNA synthesis efficiency is improved, but aptamer binding stability deteriorates
Solution Approach 1:
The system transitions from a static binding state at low temperatures to a dynamic unbinding state at elevated temperatures. The aptamer-DNA polymerase complex is stable during storage and setup, but becomes labile and dissociates when heated to reaction temperature, enabling controlled activation of DNA synthesis while maintaining compositional stability when needed
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
This approach effectively blocks DNA polymerase activity at low temperatures and fully activates it at higher temperatures, enhancing the efficiency and fidelity of DNA synthesis by ensuring precise control over aptamer activity, thereby improving DNA amplification processes like PCR.
Implementation Method 1
an endonuclease V-cleavable oligonucleotide aptamer that binds to the DNA polymerase, wherein the oligonucleotide aptamer is present in an amount effective to inhibit DNA synthesis activity of the DNA polymerase in the reaction mixture, and (iii) an endonuclease V enzymatic activity; and cleaving the aptamer by the endonuclease V enzymatic activity
Implementation Method 2
DNA polymerases are enzymes used for synthesis of DNA strands by primer extension, wherein the polymerase-catalyzed DNA synthesis may be initiated by oligonucleotide primers hybridized to a complementary template DNA
Implementation Method 3
an endonuclease V-cleavable oligonucleotide aptamer that binds to the DNA polymerase, wherein the oligonucleotide aptamer is present in an amount effective to inhibit DNA synthesis activity of the DNA polymerase
Data Source
AI summary
Provided are methods and compositions for activating oligonucleotide aptamer-deactivated DNA polymerases, comprising cleaving the aptamer by endonuclease V enzymatic activity to reduce or eliminate binding of the oligonucleotide aptamer to the DNA polymerase, thereby activating DNA synthesis activity of the DNA polymerase in a reaction mixture. Mixtures for use in methods of the invention are also provided. In some aspects, the oligonucleotide aptamer comprises one or more deoxyinosine nucleotides providing for aptamer-specific recognition and cleavage of the aptamer by the endonuclease V enzymatic activity. Exemplary oligonucleotide aptamers, mixtures and methods employing endonuclease V enzymatic activity are provided. The methods can be practiced using kits comprising a DNA polymerase-binding oligonucleotide aptamer and at least one endonuclease V enzymatic activity having oligonucleotide aptamer-specific recognition to provide for specific cleavage of the aptamer by the endonuclease V enzymatic activity.