Oligonucleotide Detection Using Spacer-Modified Capture Probes
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Solution Overview
Problem
Conventional methods for detecting and quantifying oligonucleotides lack sensitivity and specificity, particularly in distinguishing intact oligonucleotides from their metabolites, and require complex enzymatic treatments.
Innovation Solution
A method using a capture probe and an assist probe in a hybridization assay, where a spacer is inserted between the solid phase and the nucleic acid probe in the capture probe, allowing for the detection and differentiation of intact oligonucleotides from their metabolites without the need for enzymatic treatment.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional PCR methods are used for detection, then the measurement can be performed, but the sensitivity is insufficient to detect low drug concentrations and cross-reactivity with metabolites occurs
Solution Approach 1:
The detection probe is segmented into two separate functional components: a capture probe containing a first recognition sequence that binds to the 5' end region of the target oligonucleotide, and a detection probe containing a second recognition sequence that binds to the 3' end region. This segmentation allows the system to detect the full-length intact oligonucleotide while excluding metabolites that have been degraded at either end, thereby improving measurement precision and eliminating cross-reactivity.
2Measurement precision
If hybridization-ligation ELISA assay is used to distinguish intact oligonucleotide from metabolite, then specificity is improved, but the device complexity and operational complexity increase significantly
Solution Approach 1:
The invention extracts and eliminates the need for complex enzymatic ligation steps and multiple probe design requirements by using a simplified dual-probe hybridization approach. The capture probe is immobilized on a solid phase and the detection probe remains in solution, allowing for easier operation and reduced device complexity while maintaining high specificity through the requirement that both recognition sequences must bind to the same intact oligonucleotide target.
3Measurement precision
If high sensitivity detection is required for low dose treatment monitoring, then detection limit must be lowered, but conventional methods cannot achieve sufficient sensitivity
Solution Approach 1:
The capture probe acts as an intermediary that immobilizes the target oligonucleotide on a solid phase, concentrating it from the sample and enabling sensitive detection. The detection probe, labeled with a detectable marker, serves as another intermediary that provides a measurable signal. This two-stage intermediary approach amplifies the detection signal while maintaining quantitative reliability, allowing detection of low drug concentrations with high reliability.
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
The method achieves high sensitivity and specificity, enabling accurate detection and quantification of oligonucleotides while minimizing cross-reactivity with metabolites, thus meeting the stringent requirements for pharmaceutical development.
Implementation Method 1
A method using a capture probe and an assist probe in a hybridization assay
Data Source
AI summary
A method for measuring an oligonucleotide which is simpler and more sensitive and has excellent specificity and quantitative capability compared to the conventional measurement method is provided. Moreover, a method for measuring an oligonucleotide having excellent specificity which can distinguish the intact target oligonucleotide (unchanged form) and a metabolite thereof and detect the unchanged form only is provided. In a hybridization method using a capture probe and an assist probe, by inserting a spacer between a solid phase and a nucleic acid probe contained in the capture probe, it becomes possible not only to detect the target oligonucleotide in a sample but also to distinguish from a metabolite of a nucleic acid drug.