Small RNA Detection Sensor Using Target as Primer
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Solution Overview
Problem
Existing small RNA detection methods face challenges in recognizing target RNAs without ligating DNA adaptors or polyA tail RNA, leading to low recognition efficiency and non-specific primer interactions during PCR amplification, making direct sequencing of nucleotide sequences impossible.
Innovation Solution
A small RNA detection sensor with a first sensing region having amine modifications and nucleotide substitutions, such as ANA, to prevent non-specific PCR amplification and enable direct sequencing by using the target RNA as a primer for PCR amplification, allowing for high accuracy and sensitivity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If DNA adaptors are ligated to small RNAs for detection, then the detection process can proceed through reverse transcription and PCR amplification, but the recognition efficiency becomes too low and non-specific primer interactions occur during PCR
Solution Approach 1:
The invention extracts and removes the DNA adaptor ligation step from the detection workflow. Instead of ligating DNA adaptors to small RNAs, the method uses the small RNA molecules themselves as primers for PCR amplification, eliminating the source of non-specific interactions and improving recognition efficiency while maintaining detection accuracy
Solution Approach 2:
The invention inverts the conventional approach by using the target small RNA itself as the primer for PCR amplification rather than using external DNA adaptors and primers. This inversion allows the target molecule to directly initiate amplification, improving both recognition efficiency and eliminating non-specific primer interactions
2Productivity
If PCR amplification is used to amplify small RNAs, then the signal can be detected, but direct sequencing of the nucleotide sequences becomes impossible requiring additional library production
Solution Approach 1:
The invention merges the PCR amplification process with sequencing capability by designing the PCR primers to include sequencing adapter sequences. This allows the amplified products to be directly sequenced without requiring separate library production steps, reducing process complexity while maintaining high amplification efficiency
Solution Approach 2:
The PCR primers designed in this invention serve multiple functions: they initiate amplification of the small RNA targets and simultaneously provide the sequencing adapter sequences needed for direct sequencing. This multi-functionality eliminates the need for separate library preparation steps
3Productivity
If conventional PCR primers are used for small RNA amplification, then amplification can occur, but non-specific primer interactions cause inaccurate results
Solution Approach 1:
The invention employs self-service by using the small RNA target molecules themselves as the primers for their own amplification. This self-priming mechanism ensures high specificity because only the actual target molecules can initiate amplification, eliminating non-specific primer interactions while maintaining amplification capability
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
The sensor achieves high accuracy and sensitivity in detecting small RNAs, enabling direct sequencing without additional adaptors or library production, and allows for precise detection even at low concentrations.
Implementation Method 1
the small RNA detection sensor to synthesize a replication region complementary to the PCR-capable region by a DNA polymerase by using the target small RNA as a primer
Implementation Method 2
the first sensing region comprises: at least one amine modification portion that prevents non-specific PCR amplification may be caused by the self-dimer formation between two different sensors
Implementation Method 3
at least one nucleotide substitution portion for cutting the first sensing region by a nuclease, and wherein the nucleotide substitution portion comprises an ANA nucleotide substituted for a nucleotide of the first sensing region
Implementation Method 4
amplify the PCR-capable region and the replication region
Data Source
Figure 1a~1b
Figure 2(a)~2(e)
Figure 3a~3d
AI summary
The present invention may provide a small RNA detection sensor comprising: at one end thereof, a first sensing region comprising nucleotides having a sequence complementary to target small RNA; and a PCR-capable region that is coupled to the first sensing region, the small RNA detection sensor to synthesize a replication region complementary to the PCR-capable region by a DNA polymerase by using the target small RNA as a primer, and amplify the PCR-capable region and the replication region.