Nucleoside Tag Protein Detection via Antibody Binding
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Solution Overview
Problem
Current methods for tagging proteins in biological research and medical diagnostics face challenges such as protein function and localization interference, toxicity, and insufficient sensitivity and specificity, particularly with direct labeling and fluorescent molecules.
Innovation Solution
The use of nucleosides or nucleoside analogs as tags, which are attached to proteins and detected using specific binding reagents, allowing for precise localization and quantification of proteins in biological samples through methods like flow cytometry and immunohistochemistry.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If direct labelling of proteins with fluorophore dyes or fusion tags is used, then detection capability is improved, but protein function and localization are interfered with due to large tag size
Solution Approach 1:
The invention extracts the detection function from large protein tags and fluorophores, replacing them with small nucleoside analog tags (e.g., 5-iodo-2'-deoxyuridine, 5-bromo-2'-deoxyuridine) that can be incorporated into proteins during translation. These minimal tags provide detection capability while causing negligible interference with protein function and localization, directly resolving the contradiction between detection precision and protein reliability.
Solution Approach 2:
The invention changes the detection parameter from large molecular tags (fluorophores, fusion proteins) to small chemical nucleoside analogs. This parameter change in tag size and chemical nature enables detection while preserving protein behavior, as the nucleoside tags are small molecules that do not significantly alter protein structure or function compared to large fluorophore or fusion tag labels.
2Measurement precision
If fluorescent molecules such as fluorescein are used for tagging, then detection is enabled, but background fluorescence increases reducing specificity
Solution Approach 1:
The invention introduces an intermediary detection system where nucleoside analog tags (e.g., 5-iodo-2'-deoxyuridine, 5-bromo-2'-deoxyuridine) are incorporated into proteins, and then specific antibodies or binding reagents are used to detect these tags. This intermediary approach eliminates background fluorescence because the nucleoside tags themselves are non-fluorescent, and only the specific binding reagents produce detectable signals, thereby improving detection specificity.
3Measurement precision
If traditional tagging methods are used, then protein detection is achieved, but toxicity is induced or sensitivity and specificity are insufficient
Solution Approach 1:
The invention uses transient, non-persistent nucleoside analog tags (e.g., 5-iodo-2'-deoxyuridine, 5-bromo-2'-deoxyuridine) that are incorporated into proteins during translation and can be detected without long-term persistence. These tags are metabolically stable enough for detection but do not accumulate to toxic levels, providing a disposable tagging approach that maintains sensitivity and specificity while minimizing toxicity compared to traditional fluorescent proteins or chemical labels.
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 enables simultaneous tracking of multiple proteins with high sensitivity and specificity, without interfering with protein behavior, and allows for confirmation of results through chemical modification of the tags, providing a robust method for protein detection and localization.
Implementation Method 1
contacting the biological sample with a binding reagent that binds specifically to the tag
Implementation Method 2
The binding reagent may contain or be attached to a fluorescent group, which makes the binding reagent fluorescent. In this case, the location of the tagged protein in the sample is determined by obtaining an image of fluorescence emitted from the florescent group on the binding reagent.
Data Source
AI summary
Provided are methods and reagents for detecting polypeptides using nucleosides or nucleoside analogs as tags. In particular, a tagged polypeptide is contacted with a binding reagent (such as an antibody) that binds specifically to the nucleoside tag portion of the tagged polypeptide. The binding of the binding reagent to the nucleoside tag portion of the tagged polypeptide is then detected, thereby quantifying or localizing the tagged polypeptide. Provided is a variety of uses of this technology. For example, the tag specific antibodies can be presented in an array that is suitable for quantifying or characterizing a number of tagged proteins in a number of liquid samples. The technology can also be used to track a large number of tagged proteins in vivo, for example, by multiplex immunohistochemistry.