Rolling Circle Amplification for Target Molecule Detection
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Solution Overview
Problem
Current methods for detecting target molecules, such as proteins, are complex and require expensive equipment, limiting their use in simple tests or self-medication, and existing rolling circle amplification methods are inefficient for nucleic acid detection.
Innovation Solution
A method using single-stranded circular DNA, a capture oligonucleotide, and an oligonucleotide primer with aptamer sequences to form a complex with the target molecule, followed by rolling circle amplification and detection with a guanine quadruplex-binding reagent like ThT derivative, allowing for efficient detection of non-nucleic acid molecules at constant temperature.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If real-time PCR method or ELISA method is used for detecting target molecules, then detection accuracy is improved, but device complexity and cost increase
Solution Approach 1:
The patent replaces complex mechanical detection systems (PCR thermal cyclers, ELISA plate readers) with a simple isothermal rolling circle amplification system that uses DNA polymerase to generate long repetitive DNA sequences, which are then detected by a guanine quadruplex-binding reagent. This substitution maintains detection accuracy while eliminating the need for expensive temperature-cycling equipment.
Solution Approach 2:
The patent changes the reaction parameter from temperature-cycling (PCR) to isothermal conditions (constant temperature around 37-50°C). This parameter change simplifies the detection system by eliminating thermal cyclers while maintaining amplification efficiency through the rolling circle mechanism that operates optimally at constant temperature.
2Measurement precision
If real-time PCR method or ELISA method is used for detecting target molecules, then detection accuracy is improved, but usage cost increases
Solution Approach 1:
The patent employs disposable, inexpensive components: a simple oligonucleotide primer with aptamer, a circular DNA template, and a guanine quadruplex-binding reagent. These replace expensive reusable equipment like PCR machines and ELISA readers, making the test suitable for point-of-care and self-medication applications where cost-effectiveness is critical.
3Productivity
If conventional rolling circle amplification uses analyte RNA as primer, then detection is enabled, but amplification efficiency and detection efficiency are insufficient
Solution Approach 1:
The patent performs preliminary action by pre-designing a specific oligonucleotide primer containing an aptamer sequence that is complementary to the circular DNA template. This pre-configured primer is ready to initiate rolling circle amplification immediately upon target binding, eliminating the need to use the analyte RNA itself as the primer and thereby significantly improving amplification efficiency and detection sensitivity.
4Ease of operation
If simple detection methods are used, then ease of operation is improved, but detection capability for non-nucleic acid molecules is insufficient
Solution Approach 1:
The patent achieves universality by using aptamers (nucleic acid sequences) that can bind to diverse target molecules including proteins, small molecules, and other non-nucleic acid analytes. The rolling circle amplification system itself is universal and can detect any target as long as a suitable aptamer is designed, making the method highly adaptable while maintaining simplicity and ease of operation.
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 specific and sensitive detection of target molecules, including proteins, without the need for temperature cycling, making it suitable for simple and cost-effective applications in diagnostics and examinations.
Implementation Method 1
the oligonucleotide primer contains a first aptamer sequence which binds to the target molecule... and a second aptamer sequence which is linked to the 3'-side of the sequence complementary to the second region and binds to the target molecule
Implementation Method 2
performing a nucleic acid amplification reaction by rolling circle amplification based on the formation of the complex
Implementation Method 3
detecting amplified nucleic acid... using a guanine quadruplex-binding reagent like ThT derivative
Data Source
AI summary
A method of detecting a target molecule, the method comprising: forming a complex of a target molecule, a capture oligonucleotide, an oligonucleotide primer, and a single-stranded circular DNA; performing a nucleic acid amplification reaction by rolling circle amplification based on the formation of the complex; and detecting amplified nucleic acid; wherein the single-stranded circular DNA contains a first region, and a second region linked to the 3′-side of the first region, and preferably further contains a sequence complementary to a detection reagent-binding sequence; the primer contains a first aptamer sequence which binds to the target molecule, and a sequence which is linked to the 3′-side of the first aptamer sequence and is complementary to the first region of the single-stranded circular DNA; and the capture oligonucleotide contains a sequence complementary to the second region of the single-stranded circular DNA, and a second aptamer sequence which is linked to the 3′-side of the sequence complementary to the second region and binds to the target molecule.