Protein-Polynucleotide Conjugate Assay for Complex-Matrix Detection
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Solution Overview
Problem
Existing methods for detecting and quantitating intact protein-polynucleotide conjugate molecules, such as antibody-siRNA conjugates, are not compatible with complex sample matrices like serum and tissue homogenates, and often require high temperatures that can degrade proteins or rely on inefficient PCR steps.
Innovation Solution
A method using tagged triplex forming oligonucleotides (TFOs) that hybridize with polynucleotides in conjugate molecules, followed by a capture reagent that binds to either the tag or protein, and a detection reagent with a detectable label to quantify the conjugates in a sandwich-based assay.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If size exclusion chromatography is used to separate intact protein-polynucleotide conjugate molecules, then separation of conjugates from other species is improved, but the method requires high sample purity and is not compatible with complex sample matrices
Solution Approach 1:
The patent introduces a triplex-forming oligonucleotide (TFO) as an intermediary molecule that specifically binds to the polynucleotide component of the conjugate. This TFO acts as a mediator that enables selective capture and detection of intact conjugates in complex matrices without requiring high sample purity, thereby resolving the contradiction between detection accuracy and adaptability to complex samples
2Measurement precision
If PCR-based detection methods are used, then detection sensitivity is improved, but high temperatures required for PCR can result in protein degradation
Solution Approach 1:
The patent replaces the thermal cycling mechanism of PCR with a hybridization-based capture system using TFOs and sandwich assays. This substitution eliminates the need for high-temperature processing while maintaining detection sensitivity, as the TFO-polynucleotide hybridization occurs at lower temperatures that preserve protein integrity
3Measurement precision
If SDS-PAGE or LC-MS methods are used, then detection capability is improved, but time-consuming sample processing and post-assay data interpretation steps are required
Solution Approach 1:
The patent extracts and isolates only the essential detection function from complex analytical methods. By using TFO-based hybridization and sandwich assays, the method directly detects intact conjugates in a single step without requiring time-consuming sample processing, electrophoresis, or complex data interpretation procedures
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 robust detection and quantitation of protein-polynucleotide conjugates in various sample types, including complex biological samples, without protein degradation and with high sensitivity.
Implementation Method 1
contacting the sample with a triplex forming oligonucleotide (TFO) that is covalently linked to a tag under conditions that allow the TFO to hybridize to the polynucleotide in the conjugate molecule
Implementation Method 2
contacting the hybridization mixture with a surface comprising a capture reagent that specifically binds to the tag covalently linked to the TFO
Implementation Method 3
contacting the surface with a detection reagent, wherein the detection reagent comprises a detectable label coupled to a binding partner that specifically binds to the protein in the conjugate molecule; and detecting a signal from the detectable label
Data Source
AI summary
The present invention relates to methods for detecting and quantifying intact protein-polynucleotide conjugate molecules in various sample matrices. In particular, the methods utilize triplex forming oligonucleotides in combination with protein-specific binding partners to respectively detect the polynucleotide and protein components of the conjugate molecules.