Sense–Antisense Oligonucleotide Duplex Detection Without Extraction
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Solution Overview
Problem
Current methods for detecting and quantifying oligonucleotide therapeutics are limited by assay sensitivity and time-consuming extraction steps, leading to inefficiencies in drug development and increased risks in human trials due to poor pharmacokinetic understanding.
Innovation Solution
A method involving a set of probes that include sense and antisense oligonucleotide tags, which hybridize to a duplex on a support surface, followed by hybridization and immobilization, and detection using single-strand specific nucleases to quantify both strands.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional PCR-based, SEC, or LC-MS methods are used for characterizing nucleic acid therapeutics, then the assay can be performed with existing technology, but the assay sensitivity is limited and extraction steps are time-consuming
Solution Approach 1:
The method segments the oligonucleotide detection process into separate capture and detection phases. Capture probes are immobilized on a solid phase to selectively capture sense and antisense strands, while detection probes with labels are used to quantify the captured strands. This segmentation enables high sensitivity without time-consuming extraction steps by directly measuring the therapeutic oligonucleotides in biological matrices.
Solution Approach 2:
The patent introduces capture probes as intermediary molecules that bridge the target oligonucleotides and the detection system. These capture probes with specific sequences and immobilization capabilities serve as mediators to selectively bind and concentrate the sense and antisense strands, enabling sensitive detection in complex biological samples without requiring extensive extraction procedures.
2Loss of information
If quantitative assays are performed to characterize nucleic acid pharmacokinetics, then pharmacokinetic understanding is improved, but the complexity of the assay increases
Solution Approach 1:
The assay is segmented into distinct functional components: capture probes for selective binding, detection probes for quantification, and a solid phase for immobilization. This segmentation simplifies the overall assay design by separating the capture and detection functions, enabling comprehensive pharmacokinetic characterization without excessive complexity.
Solution Approach 2:
The detection probes serve multiple functions: they bind to the captured oligonucleotide strands, provide labels for signal detection, and enable quantification of both sense and antisense strands. This multi-functionality reduces the number of separate reagents and steps needed, simplifying the assay while maintaining comprehensive pharmacokinetic information.
3Loss of information
If both sense and antisense strands are quantified to understand stability and metabolic pathways, then pharmacological understanding is improved, but the assay time and resource requirements increase
Solution Approach 1:
The method merges the detection of sense and antisense strands into a single simultaneous assay. Both strands are captured and detected in the same reaction mixture using paired capture probes and detection probes, enabling parallel quantification of both strands without requiring separate assays. This merging dramatically improves throughput while providing comprehensive stability and metabolic pathway information.
Solution Approach 2:
The capture probes and detection probes are designed to simultaneously recognize and quantify both sense and antisense strands. The detection probes can bind to either strand type, and the solid phase can immobilize both capture probe types, creating a universal assay system that efficiently processes both strands in one experiment, thereby increasing productivity.
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
Provides sensitive and efficient detection and quantification of oligonucleotide strands with a limit of detection below 200 μg/mL, improving pharmacokinetic understanding and reducing animal usage in drug development.
Implementation Method 1
a sense probe that includes a first single stranded oligonucleotide tag that is complementary to at least a portion of a first capture oligonucleotide immobilized on a support surface, a sense binding portion capable of hybridizing to a nucleotide sequence of the sense strand of the oligonucleotide duplex
Implementation Method 2
contacting the hybridization mixture with a single-strand specific nuclease
Data Source
AI summary
Described herein is a method for detecting an oligonucleotide in a sample, and in particular, to a method for detecting sense and antisense strands of an oligonucleotide duplex in a sample.