Asymmetric Stem-Loop Oligomers for Multiplex Detection
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Solution Overview
Problem
Existing target capture oligomers face challenges in reducing non-specific binding to immobilized probes in the absence of target nucleic acid, which can lead to reduced sensitivity in detecting target nucleic acids, especially in multiplex methods where multiple targets are present.
Innovation Solution
The development of target capture oligomers with first and second stem segments of unequal length, forming a stem-loop structure that remains inactive without the target nucleic acid, allowing the target-binding segment to hybridize and disrupt the stem-loop, enabling specific binding to an immobilized probe only when the target is present, thereby enhancing sensitivity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If target capture oligomers are used to bind immobilized probes, then target nucleic acid can be captured, but non-specific binding occurs in the absence of target reducing sensitivity
Solution Approach 1:
The target capture oligomer employs a dynamic stem-loop structure that can transition between closed (inactive) and open (active) states. The loop region containing the immobilized probe binding sequence is sequestered in the stem structure under hybridizing conditions without target, preventing non-specific binding. When target nucleic acid is present, it binds to the loop region, causing the stem to open and exposing the binding sequence for specific immobilized probe interaction.
Solution Approach 2:
The invention changes the structural parameters of the target capture oligomer based on target presence. Without target, the oligomer maintains a compact stem-loop conformation with reduced accessibility of the binding sequence. Upon target binding, the conformational parameter changes as the stem opens, increasing accessibility and enabling specific binding to immobilized probes, thereby improving detection sensitivity.
2Reliability
If the target capture oligomer forms a stem-loop structure, then non-specific binding is reduced, but the structure must be disrupted for target binding
Solution Approach 1:
The target capture oligomer is segmented into distinct functional regions: a stem region that forms the stable structure, a loop region containing the immobilized probe binding sequence, and a target binding region. This segmentation allows the stem to provide structural stability and prevent non-specific binding, while the loop and target binding regions remain accessible for specific target recognition and immobilized probe interaction.
Solution Approach 2:
The stem-loop structure employs asymmetric design where the stem and loop regions have different sequences and structural properties. The stem region is designed to form stable intramolecular hybridization, while the loop region is configured to specifically bind target nucleic acid. This asymmetry ensures that the structure naturally opens upon target binding without requiring external disruption, simplifying the overall mechanism.
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 improves target sensitivity by ensuring that the target capture oligomers remain inactive until the target nucleic acid is bound, reducing non-specific binding and increasing the efficiency of target detection, especially in multiplex assays.
Implementation Method 1
Under hybridizing conditions in the absence of the target nucleic acid the target capture oligomer forms a stem-loop, intramolecular hybridization of the first and second stem segments forming the stem
Implementation Method 2
in the presence of the target nucleic acid, the target-binding segment hybridizes to the target nucleic acid disrupting the intramolecular hybridization of the first and second stem segments
Implementation Method 3
the first stem segment being accessible to hybridize to a complementary immobilized probe
Data Source
AI summary
The invention provides an improved stem-loop target capture oligomer and methods of use. Such a target capture oligomer has a target-binding segment forming a loop flanked by stem segments forming a stem. The stem segments are of unequal length. Such probes show little or no binding to immobilized probes in the absence of a target nucleic acid but offer good target sensitivity. The probes are particularly useful in multiplex methods of detection in which multiple target capture oligomers are present for detecting of multiple target nucleic acids (for example, detecting multiple polymorphic forms of a target gene).