Aptamer Barcode Release for Multiplex Ligand Detection
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Solution Overview
Problem
Current methods for detecting small molecule ligands in biological fluids, such as mass spectrometry and HPLC, require expensive equipment and large sample volumes, and structure-switching aptamers (SSAs) lack multiplexing capabilities and signal amplification, making them ineffective for low-abundance analytes in limited sample volumes.
Innovation Solution
A system using ligand-sensing complexes with ligand-binding oligonucleotides (LBOs) hybridized to barcoded short-release oligonucleotides (SROs) that undergo conformational changes upon ligand binding, releasing unique barcode sequences for detection, enabling multiplexed analysis and improved sensitivity and dynamic range.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If structure-switching aptamers (SSAs) are used for ligand detection, then specific binding and conformational change detection are achieved, but multiplexing capability is lost because every SSA has the same readout
Solution Approach 1:
The system divides the readout function into separate barcode sequences that are released upon ligand binding. Each SSA is assigned a unique barcode, allowing multiple SSAs to be multiplexed in a single assay while maintaining readout distinguishability through sequence identification rather than shared fluorescent signals.
Solution Approach 2:
The patent introduces barcode sequences as intermediary elements that mediate between the ligand-binding event and the final readout. The barcode acts as an information carrier that distinguishes which SSA detected which ligand, enabling multiplexing without signal cross-contamination.
2Measurement precision
If conventional ligand detection methods (mass spectrometry, HPLC) are used, then detection accuracy is improved, but sample volume requirements increase and equipment costs rise
Solution Approach 1:
The system uses barcode sequence copying and amplification to detect ligand binding events. Instead of directly measuring ligand mass or chromatographic properties, the method copies the barcode information through nucleic acid amplification techniques, enabling high sensitivity detection with minimal sample volume and standard laboratory equipment.
3Reliability
If structure-switching aptamers are used for low-abundance analyte detection, then binding specificity is maintained, but signal amplification is prevented due to identical readouts
Solution Approach 1:
The system implements feedback through barcode amplification and sequencing. The released barcode sequences serve as feedback signals that can be amplified and detected with high sensitivity, allowing the system to detect low-abundance ligands while maintaining the binding specificity of the original SSA-Ligand interaction.
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 the simultaneous detection of multiple ligands in a single assay volume by releasing distinct barcode sequences, allowing for sensitive and efficient analysis of low-abundance analytes without the need for large sample volumes or expensive equipment.
Implementation Method 1
binding of the target ligand to the LBO drives a conformational change triggering release of the barcoded SRO from the LBO/SRO pairing
Implementation Method 2
LBO hybridized to a corresponding barcoded short-release oligonucleotide (SRO) to form an LBO/SRO pairing, the LBO comprising a ligand-binding region that specifically binds to the target ligand and an SRO hybridization region sufficiently complementary to a corresponding LBO hybridization region
Data Source
AI summary
Described herein are multiplexable aptamer-based systems and methods for detecting target ligands in a fluid sample. More specifically, described herein are ligand-sensing complexes comprising a ligand-binding oligonucleotide (LBO) hybridized to a corresponding short-release oligonucleotide (SRO) such that binding of a target ligand to the LBO drives a conformational change triggering release of a barcoded SRO or LBO. The released barcode, which comprises a sequence that is informative with respect to the target ligand bound, may then be captured, amplified and/or sequenced as a readout for the presence/concentration of the target ligand in the fluid sample. Also described herein is a method for preparing ligand-sensing complexes with error-free LBO/SRO pairing, as well as a method for improving the sensitivity and/or dynamic range of aptamer-based detection systems.