Oligonucleotide Detection via Surface-Charged Probes and AEX-HPLC
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Solution Overview
Problem
Current methods for detecting and quantifying small regulatory RNAs like miRNAs in biological samples are time-consuming, expensive, and lack sensitivity, particularly in high-throughput applications, due to the need for intensive sample preparation and amplification in RT-PCR, and existing chromatographic methods struggle with separating oligonucleotides of similar length.
Innovation Solution
A method employing fluorescently labeled, chemically modified detection molecules with different surface charges in combination with anion-exchange high performance liquid chromatography (AEX-HPLC) allows for the simultaneous detection and separation of multiple oligonucleotides of equal length by altering their overall surface charges upon hybridization, enabling improved resolution and quantification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If RT-PCR methods are used for detection and quantification of small regulatory RNAs, then detection sensitivity and quantification capability are improved, but the method becomes time-consuming and expensive due to intensive sample preparation and amplification requirements
Solution Approach 1:
The invention extracts and eliminates the time-consuming amplification step from the detection workflow by using direct hybridization-based detection. Detection molecules with different surface charges are directly hybridized to target oligonucleotides in the sample, and the hybrids are separated and detected without requiring prior PCR amplification, thus maintaining detection sensitivity while dramatically reducing time and cost
Solution Approach 2:
The invention changes the physical-chemical parameters of the detection system by using detection molecules with different surface charges (positive, negative, neutral) instead of relying on amplification. This parameter change enables direct detection of low-abundance targets through selective hybridization and charge-based separation, eliminating the need for time-consuming amplification cycles while maintaining quantification capability
2Manufacturing precision
If conventional chromatographic methods are used for oligonucleotide separation, then separation capability is achieved, but the method struggles with separating oligonucleotides of similar length
Solution Approach 1:
The invention applies local quality by modifying detection molecules with specific surface charge characteristics tailored to each target oligonucleotide. Each detection molecule is designed with a specific charge property (positive, negative, or neutral) that creates a unique hybrid complex with distinct chromatographic behavior, enabling resolution of oligonucleotides of similar length through localized charge differentiation rather than relying solely on length differences
Solution Approach 2:
The invention uses composite detection molecules that combine hybridization specificity (nucleic acid sequence complementarity) with charge-based separation properties. These composite molecules integrate both recognition functionality and separation functionality, allowing simultaneous specific binding to target oligonucleotides and differential separation based on the composite charge characteristics of the detection molecule-oligonucleotide hybrid
3Productivity
If multiple oligonucleotides are detected in parallel, then throughput and efficiency are improved, but the complexity of the detection system increases
Solution Approach 1:
The invention achieves universality by using a single chromatographic system with a universal separation mechanism (anion-exchange) that can handle multiple detection molecules with different surface charges in parallel. The same column and elution conditions separate all hybrid complexes based on their charge characteristics, enabling multiplex detection without requiring multiple specialized systems or complex instrumentation
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 rapid, sensitive, and reliable quantification of multiple small regulatory RNAs or DNA molecules in a single biological sample, overcoming the limitations of existing methods by allowing parallel detection and reducing the need for extensive sample preparation.
Implementation Method 1
forming a hybridization mixture by contacting the biological sample with at least two detection molecules complementary to the at least two distinct oligonucleotides
Implementation Method 2
separating the detection molecules hybridized to the at least two distinct oligonucleotides of equal length from the moiety of non-hybridized detection molecules by anion exchange high performance liquid chromatography
Implementation Method 3
each of the detection molecules is labelled with at least one fluorescent moiety
Data Source
AI summary
Provided herein is a method for detecting at least two distinct oligonucleotides of equal length in parallel from one biological sample, comprising the steps of providing a biological sample containing or suspected of containing oligonucleotides of interest; forming a hybridization mixture using at least two fluorescently labelled detection molecules with different surface charges; separating the detection molecules hybridized to the oligonucleotides by anion exchange HPLC; and detecting the hybridized detection molecule-oligonucleotide moieties by quantitative fluorescence readout. In a further aspect, a kit comprising at least two detection molecules is provided. In another aspect, provided herein is the use of at least two detection molecules with different surface charges for quantitatively detecting at least two distinct oligonucleotides of equal length in parallel from one biological sample.