Single-Base RNA Modification Detection via Enzymatic Hindrance
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Solution Overview
Problem
Current methods for detecting N6-methyladenosine (m6A) modifications in RNA lack single-base resolution and specificity, limiting their ability to quantify and identify specific m6A sites effectively, particularly in RNA transcripts.
Innovation Solution
A method involving the design of specific DNA probes that anneal to RNA sequences surrounding the target site, using PCR amplification and qPCR for detection, which exploits the hindrance of m6A marks on DNA polymerase elongation and ligase activity, allowing for selective detection and quantification of m6A modifications at single-base resolution.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If m6A-IP or antibody-based methods are used for detection, then transcriptomic-wide information can be obtained, but measurement precision and single-base resolution are insufficient
Solution Approach 1:
The invention divides the RNA molecule into segments by using two probes that bind to different regions (upstream and downstream of the target site). The probes are elongated and ligated only when the RNA is intact and properly structured, enabling segment-specific detection at single-base resolution without requiring complex whole-transcriptome analysis methods
Solution Approach 2:
The invention introduces DNA probes as intermediary molecules that bridge the detection process. These probes bind to RNA, undergo elongation by DNA polymerase, and are ligated by ligase to form a detectable product. This intermediary system enables precise detection at single-base resolution while maintaining methodological simplicity
2Measurement precision
If SCARLET method is used for single-base detection, then quantitative detection at single-base resolution is achieved, but time consumption and radioactive labeling requirements increase
Solution Approach 1:
The invention replaces expensive and hazardous radioactive labels with inexpensive, non-radioactive fluorescent labels or other detectable markers. The probes are designed as disposable oligonucleotides that are elongated and ligated in a single reaction, eliminating the need for time-consuming radioactive labeling steps while maintaining single-base resolution
Solution Approach 2:
The invention replaces the complex enzymatic and radioactive labeling system of SCARLET with a simplified DNA polymerase elongation and ligase ligation system. This mechanical-enzymatic substitution eliminates radioactive materials and reduces detection time while preserving single-base resolution capability
3Measurement precision
If PA-m6A-seq or miCLIP methods are used, then higher resolution is achieved, but specificity and crosslinking yields remain problematic
Solution Approach 1:
The invention extracts and eliminates the problematic UV crosslinking step from the detection process. Instead of relying on antibody-RNA crosslinking under UV exposure, the method uses direct hybridization of DNA probes to RNA followed by enzymatic elongation and ligation, thereby removing the source of low specificity and low crosslinking yields while maintaining high resolution
Solution Approach 2:
The invention changes the detection parameters from antibody-based crosslinking to DNA probe-based hybridization and enzymatic processing. This parameter change from chemical crosslinking to sequence-specific binding and enzymatic ligation improves both specificity and reliability while maintaining single-base resolution
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 precise and efficient detection of m6A sites in RNA at single-base resolution, overcoming the limitations of existing methods by providing high sensitivity and specificity without the need for radioactive labeling.
Implementation Method 1
elongating the down probe Px2 through a DNA polymerase to obtain an elongated down probe Px2
Implementation Method 2
ligating the up probe Px1 and the elongated down probe Px2 through a ligase to obtain a SELECT product
Implementation Method 3
exploits the hindrance of m6A marks on DNA polymerase elongation and ligase activity
Data Source
AI summary
Provided is a method for detecting the chemical modification of a target RNA site X, comprising the steps as follows: (1) acquiring an RNA sample and selecting in the RNA sample a target RNA segment comprising the target RNA site X; (2) SELECT; (3) PCR amplification; (4) comprising the PCR cycle threshold value with a reference PCR cycle threshold value, or comparing the PCR amplification product quantity with a reference PCR amplification product quantity, so as to determine whether there is a target chemical modification in the target RNA site X. Further provided are a method for identifying a substrate target site of RNA modification enzyme or RNA demodification enzyme and a method for quantifying an RNA modification rate in a transcript.