Cleavable Nucleic Acid Linkers for Stoichiometric Protein Quantification
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Solution Overview
Problem
Existing protein quantification methods are time-consuming, laborious, and inaccurate due to limited sensitivity, cellular heterogeneity, and variability in antibody detection, lacking the ability for non-destructive, single-cell, and real-time protein quantification.
Innovation Solution
A cleavable nucleic acid linker (PQR) is integrated into a nucleic acid molecule encoding a protein of interest and a reporter protein, allowing for cleavage during translation to quantify the protein of interest by measuring the reporter protein's signal.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional protein assays and Western blots are used for protein quantification, then protein amounts can be measured, but the methods are time-consuming, laborious, and require large quantities of isolated cells due to limited sensitivity
Solution Approach 1:
The patent introduces a reporter protein as an intermediary that is co-translated with the protein of interest in a stoichiometric ratio. The reporter protein serves as a measurable surrogate that amplifies the detection signal, enabling sensitive quantification of the protein of interest without requiring direct measurement of the target protein itself. This mediator approach allows single-cell resolution and real-time measurement.
Solution Approach 2:
The invention creates a copy of the protein expression information through the reporter protein. Instead of directly measuring the protein of interest, the system generates a corresponding copy (reporter protein) that carries the same quantitative information but is easier to detect with high sensitivity. The reporter protein acts as a informational copy that can be measured to infer the amount of the target protein.
2Measurement precision
If traditional protein assays are used, then protein quantification can be performed, but cellular resolution is limited due to heterogeneous cell populations and destructive processing
Solution Approach 1:
The patent segments the measurement process to operate at the single-cell level rather than requiring bulk heterogeneous cell populations. By expressing the reporter protein within individual cells, the system enables cell-by-cell quantification, resolving the heterogeneity issue. The cleavable linker further segments the fusion protein into separate components after measurement, allowing non-destructive assessment.
Solution Approach 2:
The reporter protein system is self-contained within each cell, requiring no external destructive processing for measurement. The fluorescence or other detectable signal from the reporter protein can be measured while the cell remains viable, enabling live-cell tracking and repeated measurements over time without cell lysis or destruction.
3Measurement precision
If immuno-detection methods are used for protein quantification, then protein amounts can be measured, but inaccuracies occur due to antibody variability in avidity, affinity, and cross-reactivity
Solution Approach 1:
The patent replaces complex, variable antibody detection systems with a simpler, more reliable reporter protein system. The reporter protein provides an intrinsic, consistent signal that does not depend on antibody properties. This disposable-like approach uses the reporter protein's inherent detectability (fluorescence, enzymatic activity) rather than requiring external antibody reagents with variable characteristics.
Solution Approach 2:
The invention substitutes the biochemical antibody-antigen recognition system with a more direct detection mechanism based on reporter protein signals. Instead of relying on mechanical/chemical interactions between antibodies and epitopes (which introduce variability), the system uses the reporter protein's intrinsic physical properties (fluorescence emission, enzymatic conversion) for detection, eliminating antibody-related inaccuracies.
4Measurement precision
If housekeeping proteins are used for normalization, then protein quantification can be standardized, but inaccuracies arise from differences in expression across conditions and cellular heterogeneity
Solution Approach 1:
The patent merges the quantification of the protein of interest and the normalization reference into a single measurement system. The reporter protein and the protein of interest are co-expressed in a fixed stoichiometric ratio from the same mRNA transcript, ensuring that both are subject to identical regulatory conditions. This merging eliminates the need for separate housekeeping protein measurements and their associated normalization errors.
Solution Approach 2:
The invention changes the normalization parameter from external housekeeping protein expression levels to the intrinsic stoichiometric ratio between the reporter protein and the protein of interest. Since both proteins are produced from the same translational event, their ratio remains constant regardless of cellular condition changes, providing a more stable and accurate normalization reference that adapts to different experimental conditions.
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 sensitive, non-destructive, and real-time protein quantification at single-cell resolution with improved accuracy by using a stoichiometric ratio of reporter and protein of interest, overcoming limitations of traditional methods.
Implementation Method 1
the PQR linker is capable of being cleaved during the translation of a messenger RNA to quantify a protein of interest
Data Source
AI summary
The present disclosure concerns a Protein Quantitation Reporter (PQR) linker which is capable of being cleaved during the translation of a messenger RNA to quantify a protein of interest. The PQR linker can encode a peptide of SEQ ID NO: 23 and have the nucleic acid sequence of SEQ ID NO: 25. The PQR linker is located in a nucleic acid molecule encoding a poly-protein between a reporter protein and the protein of interest. While the messenger RNA encoding the poly-protein is being translated, the presence of the PQR linker causes cleavage of the poly-protein and consequently the release at a stoichiometric ratio of the reporter protein and the protein of interest. The signal associated with the cleaved reporter protein can be measured to estimate or quantify the protein of interest.