Genetically Encoded FRET ATP Sensor for Live Cell Kinetic Monitoring
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Solution Overview
Problem
Standard approaches to monitoring drug-induced metabolic perturbations are limited by endpoint assays that provide population-based measurements with limited kinetic information, lacking the ability to conduct direct, automated live cell analysis of cellular ATP levels.
Innovation Solution
Development of genetically encoded ATP sensors in the form of fusion proteins that utilize fluorescence resonance energy transfer (FRET) to detect ATP levels in living cells, comprising a FRET acceptor and donor polypeptides with an ATP binding protein, allowing for real-time measurement of ATP levels.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If endpoint assays are used for monitoring drug-induced metabolic perturbations, then population-based measurements can be obtained, but kinetic information is limited
Solution Approach 1:
The patent replaces traditional endpoint chemical assays with a genetically encoded fluorescent sensor system that utilizes fluorescence resonance energy transfer (FRET) to detect ATP levels. This optical measurement system enables continuous, real-time monitoring of intracellular ATP dynamics, providing kinetic information that was previously inaccessible through standard endpoint assays.
Solution Approach 2:
The invention introduces a genetically encoded ATP sensor as an intermediary molecule that binds to ATP and transduces the chemical signal into a fluorescent signal. This sensor acts as a mediator between the intracellular ATP pool and the external detection system, enabling non-invasive, continuous measurement of ATP levels without disrupting cellular metabolism.
2Ease of operation
If traditional endpoint assays are used, then automated live cell analysis can be avoided, but direct real-time monitoring of ATP levels is not possible
Solution Approach 1:
The genetically encoded ATP sensor is expressed within the cell itself, making the cell autonomous in its sensing capability. The sensor continuously monitors ATP levels and generates fluorescent signals that can be detected externally, eliminating the need for cell lysis or external reagent addition at each measurement time point. This self-service approach enables automated, high-throughput live cell analysis.
3Extent of automation
If genetically encoded ATP sensors are implemented, then direct automated live cell analysis is enabled, but device complexity increases
Solution Approach 1:
The patent combines multiple functional domains into a single genetically encoded fusion protein: the ATP-binding domain (from the P2X7 receptor), the FRET donor (cyan fluorescent protein), and the FRET acceptor (yellow fluorescent protein), connected by flexible linkers. This merged construct simplifies the overall system by integrating the sensing, signal transduction, and reporting functions into one molecular entity that can be expressed as a single gene product.
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
Enables direct, automated, and continuous monitoring of ATP levels in living cells, providing kinetic information and enabling the assessment of metabolic perturbations induced by drugs or other substances.
Implementation Method 1
fusion proteins are disclosed, comprising a polypeptide of genus X1-X2-X3-X4-X5, wherein: one of X1 and X5 comprises a fluorescence resonance energy transfer (FRET) acceptor polypeptide having an acceptor excitation wavelength and FRET emission wavelength, and the other of X1 and X5 comprises a FRET donor polypeptide having a donor excitation wavelength and a donor emission wavelength
Data Source
AI summary
Disclosed herein are ATP biosensor fusion proteins and their use for assaying ATP levels in cells.