Nucleic Acid Detection Using Oligonucleotide Conformational Switching
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Solution Overview
Problem
Existing nucleic acid detection methods face challenges in accurately determining the concentration and degree of match of specific nucleic acids in a sample with a reference sequence, particularly at high concentrations, leading to underestimation and false negative results due to the requirement for specific nucleic acids to hybridize with multiple oligonucleotides, causing occupancy issues and reduced measurable changes.
Innovation Solution
A method and kit utilizing a first probe with a first oligonucleotide and a second oligonucleotide that is partially complementary to the first oligonucleotide or its adapter, allowing specific nucleic acids to hybridize and activate the second oligonucleotide, which then undergoes a conformation change to facilitate binding and produce a measurable change without needing to hybridize with the first oligonucleotide, thereby avoiding occupancy issues and enabling accurate concentration and match determination.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If specific nucleic acids are required to hybridize with multiple oligonucleotides (first and second) to produce a measurable change, then the detection method can provide specific signals, but at high concentrations the specific nucleic acids become occupied and cannot bind to both oligonucleotides, leading to underestimation and false negatives
Solution Approach 1:
The detection system is divided into two independent functional modules: (1) a first oligonucleotide probe that binds to the target nucleic acid to form a hybrid, and (2) a second oligonucleotide that is partially complementary to the first oligonucleotide or its adapter. This segmentation allows the target to be detected through the formation of a measurable complex without requiring the target to simultaneously bind multiple oligonucleotides, thereby eliminating the occupancy problem at high concentrations.
Solution Approach 2:
The first oligonucleotide acts as an intermediary between the target nucleic acid and the detection system. The target hybridizes with the first oligonucleotide, and the second oligonucleotide then binds to the first oligonucleotide or its adapter, creating a measurable complex. This intermediary mechanism allows indirect detection that avoids the limitation of requiring direct simultaneous binding of the target to multiple oligonucleotides.
2Adaptability or versatility
If the second oligonucleotide is made partially complementary to the first oligonucleotide or its adapter, then the system can detect the presence of specific nucleic acids through hybridization and conformational changes, but the complexity of the system increases
Solution Approach 1:
The first oligonucleotide adapter serves multiple functions: it can be used to extend the first oligonucleotide sequence, provide an additional binding site for the second oligonucleotide, and enable flexible system design. This multi-functionality allows the same adapter structure to support various detection configurations without increasing overall system complexity.
Solution Approach 2:
The second oligonucleotide is designed to be partially complementary to the first oligonucleotide or its adapter rather than fully complementary. This partial complementarity is sufficient to enable specific binding and conformational changes for detection purposes, while avoiding the need for complex fully complementary sequences, thereby simplifying the system design.
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 allows for improved detection of nucleic acid concentration and match degree, even at high concentrations, by eliminating underestimation and false negatives, as the activation of the second oligonucleotide enables direct binding between probes, enhancing sensitivity and accuracy.
Implementation Method 1
specific nucleic acid contained in the sample to be examined hybridises with the second oligonucleotide and thereby activates the second oligonucleotide
Data Source
AI summary
Method for the determination of the concentration of a type of specific nucleic acids (10) in a sample, wherein the sequences of the specific nucleic acid (10) at least partially match a reference sequence, and/or for the determination of the degree of the match of the sequences of specific nucleic acids (10) of a type in a sample (9) and a reference sequence, comprising the following steps: providing a first probe (1), which possesses a first oligonucleotide (3) on its surface; providing a second oligonucleotide (7), which is partially complementary to the first oligonucleotide (3) or to a first oligonucleotide adapter (20), which is partially complementary to the first oligonucleotide (3), and wherein the second oligonucleotide (7) is partially complementary to the reference sequence; combining of the first probe (1) and the second oligonucleotide (7) and—if applicable—the first oligonucleotide adapter (20) with the sample (9), wherein a specific nucleic acid (10) contained in the sample (9) can hybridize with the second oligonucleotide (7) and the second oligonucleotide (7) is activated by the hybridization.