Yeast BRET Telomere Assay for High-Throughput Length Screening
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Solution Overview
Problem
Current methods for measuring telomere length, such as TRF analysis and nanopore sequencing, are not suitable for high-throughput applications due to their time-consuming and material-intensive nature, hindering efficient screening for substances affecting telomere length in the context of aging and cancer research.
Innovation Solution
A high-throughput method utilizing yeast cells genetically engineered with telomere-binding proteins Rap1 and Rif2 fused with bioluminescence and fluorescence markers to measure telomere length via bioluminescence resonance energy transfer (BRET), allowing for rapid and cost-effective quantification of telomere length and identification of substances influencing telomere dynamics.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional methods like TRF analysis or nanopore sequencing are used to measure telomere length, then measurement precision is achieved, but productivity is significantly reduced due to time-consuming and material-intensive procedures
Solution Approach 1:
The patent replaces traditional mechanical/biochemical measurement systems (TRF analysis requiring radioactive labeling and gel electrophoresis, or nanopore sequencing requiring complex library preparation) with an optical energy transfer system. BRET (bioluminescence resonance energy transfer) allows telomere length measurement through non-contact optical detection, eliminating the need for radioactive materials, complex separation procedures, and extensive sample preparation, thereby enabling high-throughput screening while maintaining measurement precision
Solution Approach 2:
The patent changes the measurement parameter from direct physical measurement of DNA fragments (in TRF analysis) or sequencing reads (in nanopore sequencing) to an optical signal ratio. By measuring the ratio of bioluminescence to fluorescence intensity, the system translates telomere length into a detectable optical parameter that can be rapidly quantified in thousands of samples simultaneously, dramatically improving productivity without sacrificing precision
2Measurement precision
If traditional telomere length measurement methods are used, then accurate telomere length determination is possible, but loss of time increases due to complex procedures and long measurement cycles
Solution Approach 1:
The patent implements preliminary action by genetically engineering yeast cells to constitutively express telomere-binding fusion proteins (Rap1-YFP and Rif2-RLuc) that are always present and ready for measurement. This eliminates the need for temporary transfection, protein purification, or complex sample preparation before measurement. The cells are pre-prepared with the measurement system integrated into their genome, allowing immediate BRET measurement upon addition of substrate, thus dramatically reducing measurement time while maintaining accuracy
Solution Approach 2:
The patent extracts the measurement procedure from complex multi-step protocols (DNA extraction, fragmentation, labeling, electrophoresis in TRF analysis; or library preparation, sequencing, and bioinformatics in nanopore sequencing) and condenses it into a single-step optical measurement. By taking out only the essential detection function and implementing it through BRET in living cells, the patent reduces measurement time from days to minutes while preserving telomere length determination accuracy
3Reliability
If conventional telomere measurement approaches are employed, then reliable telomere length data is obtained, but cost increases due to expensive reagents and material-intensive procedures
Solution Approach 1:
The patent employs cheap, short-living bioluminescent substrate (coelenterazine or similar) that can be added to the assay and does not require recovery or reuse. This substrate is significantly cheaper than radioactive isotopes used in TRF analysis or the specialized reagents required for nanopore sequencing library preparation. The substrate is consumed in the reaction but enables reliable measurement without the need for expensive, reusable equipment or materials, reducing overall assay cost while maintaining reliability
Solution Approach 2:
The patent implements self-service by using the yeast cells themselves to produce and maintain the telomere-binding fusion proteins through their own genomic integration. The cells continuously express Rap1-YFP and Rif2-RLuc without requiring external addition of proteins or reagents. This eliminates the need for expensive purified proteins, antibodies, or complex buffer systems required in traditional methods, allowing reliable telomere measurement with minimal reagent consumption and lower cost
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 simple, time- and cost-effective determination of telomere length and identification of substances affecting telomere dynamics, suitable for high-throughput screening and applicable in aging and cancer research.
Implementation Method 1
Rif2 is fused with Renilla reniformis-luciferase (RLuc), which is known to emit light with maximum intensity at a wavelength of about 481 nm after conversion of the substrate coelentarazine
Implementation Method 2
increased binding of these proteins within the telomeric protein complex results in increased bioluminescence resonance energy transfer (BRET)
Implementation Method 3
Rap1 fused with a yellow fluorescent protein (YFP) acts, thereby, as an energy acceptor and emits light with a maximum intensity at a wavelength of about 527 nm when interacting with Rif1-RLuc
Data Source
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AI summary
The present invention relates to a method for determining telomere length. Further, the present invention relates to a method for identifying substances effecting telomere length. Furthermore, the present invention relates to a yeast cell which can be used in the above methods.