3′P RNA Fragment Detection via Phosphorylated Adapter Ligation
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Solution Overview
Problem
Current methods lack reliability, simplicity, and accuracy for effectively screening and quantifying 3′P RNA fragments, hindering their utilization in disease diagnosis, prognosis, and therapy monitoring.
Innovation Solution
A comprehensive method involving the Dart-RNAseq analysis and 3′P-qPCR assay for identifying and quantifying 3′P RNA fragments, which includes 5′ phosphorylation, ligation with an RNA-based adapter, intra-molecular circularization, reverse transcription, and PCR amplification, followed by targeted qPCR amplification and analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If complex and time consuming approaches are used for detecting 3′P RNA, then measurement precision may be improved, but productivity and ease of operation deteriorate
Solution Approach 1:
The patent employs an intermediary approach by using 5′ phosphorylated adapters that can ligate to 3′P RNA fragments. This adapter serves as a mediator to convert the detection of 3′P RNA into a detectable signal through sequencing, enabling accurate identification without complex direct detection methods. The adapter facilitates the interaction between the 3′P RNA and the sequencing system, resolving the contradiction between precision and productivity.
Solution Approach 2:
The patent replaces complex mechanical detection systems with a biochemical approach using enzymatic ligation and sequencing. Instead of using complex physical detection mechanisms, the invention uses molecular biology tools (phosphorylation, ligation, reverse transcription) to enable accurate detection of 3′P RNA fragments, thereby improving measurement precision while maintaining operational simplicity and throughput.
2Measurement precision
If complex and time consuming approaches are used for detecting 3′P RNA, then measurement precision may be improved, but ease of operation deteriorates
Solution Approach 1:
The patent segments the detection process into distinct modular steps: (1) 5′ phosphorylation of adapters, (2) ligation to 3′P RNA, (3) self-ligating circularization, (4) reverse transcription, and (5) PCR amplification. Each step is a simple, well-defined operation that can be performed independently. This segmentation makes the overall complex task manageable and operationally simple while maintaining high quantification accuracy through controlled conditions at each stage.
Solution Approach 2:
The patent utilizes parameter changes in the biochemical reactions to optimize detection. By controlling conditions such as temperature, pH, and reagent concentrations at each step, the method achieves high quantification accuracy. The parameters are adjusted to favor specific reactions (e.g., ligation efficiency, circularization kinetics), making the process operationally simple while maintaining precision through standardized protocol parameters.
3Ease of operation
If current detection methods relying on 3′OH group are used, then ease of operation is maintained, but reliability and measurement precision deteriorate
Solution Approach 1:
The patent inverts the conventional detection approach by targeting the 5′ end phosphorylation status rather than the 3′OH group. Instead of detecting 3′P RNA through its 3′ phosphate group directly, the method phosphorylates the 5′ end of adapters and uses this modified 5′ end for ligation and detection. This inversion maintains operational simplicity while significantly improving reliability, as the 5′ phosphate modification provides a more stable and detectable marker for 3′P RNA identification.
Solution Approach 2:
The patent introduces an intermediary adapter molecule that bridges the gap between the 3′P RNA and the detection system. The adapter, modified at its 5′ end with a phosphate group, serves as an intermediary that ligates to the 3′P RNA and carries the detectable signal. This intermediary approach maintains the simplicity of the procedure while improving reliability, as the adapter mediates the interaction and provides a more robust detection target than the 3′OH group alone.
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 more accurate and reliable diagnostic, prognostic, therapy monitoring, and outcome prediction assessments by effectively identifying and quantifying 3′P RNA fragments as molecular markers of disease.
Implementation Method 1
phosphorylating the 5′ end of the at least one 3′P RNA contained in the biological sample
Implementation Method 2
ligating the 3′ end of the at least one phosphorylated RNA fragment to the 5′ end of an RNA-based adapter
Implementation Method 3
self-ligating the at least one first ligation product to form at least one circular RNA molecule
Implementation Method 4
performing a reverse transcription of the at least one circular RNA molecule obtaining at least one single strand cDNA molecule
Implementation Method 5
performing a PCR amplification of the at least one single strand cDNA molecule
Data Source
AI summary
A method for identifying at least one RNA fragment comprising a 3′ phosphate or 2′/3′ cyclic phosphate as a molecular marker of a disease contained in a biological sample of a subject suffering from the disease and a kit for implementing the method.