Nucleic Acid Ligation With ATP Regeneration From AMP
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Solution Overview
Problem
Existing nucleic acid ligation reactions rely heavily on expensive ATP, requiring high concentrations that are unsustainable and costly, leading to inefficiencies and cofactor by-product inhibition.
Innovation Solution
A biocatalytic ligation method utilizing a fusion polypeptide comprising a polyphosphate kinase (PPK) domain and an ATP-dependent nucleic acid ligase domain, which regenerates ATP from AMP, allowing complete ligation with sub-stoichiometric ATP/AMP concentrations, reducing costs and environmental impact.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If high concentrations of ATP are used to achieve complete ligation, then ligation efficiency is improved, but process cost and environmental impact worsen
Solution Approach 1:
The patent implements ATP regeneration by recovering and recycling AMP produced during ligation back into ATP through a coupled kinase reaction. This allows sub-stoichiometric ATP concentrations to sustain complete ligation, directly resolving the contradiction between high ligation efficiency and excessive ATP consumption.
Solution Approach 2:
The dual-enzyme system maintains continuous ATP availability by immediately regenerating ATP from AMP as it is consumed during ligation. This continuous regeneration cycle eliminates the need for high initial ATP concentrations while maintaining sustained ligation activity throughout the reaction.
2Reliability
If excess ATP is used to overcome cofactor by-product inhibition, then ligation completeness is improved, but process sustainability worsens
Solution Approach 1:
Instead of discarding AMP as waste that causes inhibition, the system recovers it by converting AMP back to ATP through the kinase reaction. This eliminates the harmful accumulation of by-products while avoiding the waste of energy-rich ATP, simultaneously achieving ligation completeness and sustainability.
Solution Approach 2:
The patent converts the harmful effect of AMP accumulation (cofactor by-product inhibition) into a beneficial cycle by using the same AMP as substrate for ATP regeneration. The apparent waste product becomes the原料 for sustaining the reaction, transforming a harmful factor into a useful resource.
3Productivity
If high ATP concentrations are required for complete ligation, then ligation efficiency is improved, but process cost worsens
Solution Approach 1:
The ATP regeneration system recovers AMP and converts it back to ATP in situ, dramatically reducing the amount of expensive ATP that needs to be supplied externally. This makes the process economically viable by eliminating the need for large quantities of costly cofactor while maintaining high ligation efficiency.
Solution Approach 2:
The reaction system becomes self-sufficient for its cofactor needs through the coupled kinase reaction that autonomously regenerates ATP from AMP using polyphosphate as phosphate donor. This self-service capability eliminates dependence on external ATP supplementation, reducing both cost and 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 method achieves complete ligation of oligonucleotide fragments efficiently and sustainably, lowering costs and improving process efficiency by recycling ATP, thus overcoming the limitations of traditional methods.
Implementation Method 1
a biocatalytic ligation method utilizing a fusion polypeptide comprising a polyphosphate kinase (PPK) domain and an ATP-dependent nucleic acid ligase domain, which regenerates ATP from AMP
Implementation Method 2
The method achieves complete ligation of oligonucleotide fragments efficiently and sustainably, lowering costs and improving process efficiency by recycling ATP
Data Source
AI summary
The present disclosure relates to biocatalytic ligation methods for producing oligonucleotides; and to fusion polypeptides for use in said methods. In particular, the present disclosure relates to biocatalytic ligation methods incorporating ATP regeneration and to fusion polypeptides comprising a polyphosphate kinase domain and an ATP-dependent nucleic acid ligase domain.