Antibody-Based m6A Transcriptome Characterization via Affinity Enrichment

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Solution Overview

Problem

Current methods lack the capability to effectively characterize and detect modified bases in RNA, particularly N6-methyladenosine (m6A), which are widespread but poorly understood, due to the limitations of existing detection techniques.

Innovation Solution

A method involving the use of antibodies specific to modified bases, coupled with affinity enrichment and next-generation sequencing, allows for the characterization of modified base sites in the transcriptome by isolating and sequencing RNA transcripts that bind to these antibodies, providing a high-throughput approach to identify m6A sites.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If standard hybridization or sequencing-based methods are used to detect modified bases, then the methods are simple and widely applicable, but they cannot detect modified bases that do not alter base pairing ability (such as m6A)

Engineering Contradiction:

Improvedetectability of modified basesVSAvoidaccuracy of modified base detection

Core Design Contradiction:

Adaptability or versatilityVSReliability

Solution Approach 1:

The patent introduces an intermediary substance - an antibody specific to the modified base (m6A) - that mediates the detection process. The antibody binds specifically to the modified base, enabling indirect detection through immunoprecipitation followed by sequencing. This resolves the contradiction by providing a reliable detection method for modified bases that do not alter base pairing, while maintaining simplicity through a standardized workflow.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Reliability

If antibody-based immunoprecipitation is used to isolate modified base-containing transcripts, then detection reliability improves, but the complexity of the method increases

Engineering Contradiction:

Improveaccuracy of modified base detectionVSAvoidcomplexity of detection method

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The patent segments the detection process into distinct, manageable steps: (1) immunoprecipitation to isolate modified base-containing transcripts, (2) library preparation, and (3) high-throughput sequencing. This segmentation reduces the overall complexity by breaking down the complex detection task into modular components, each optimized for reliability while maintaining procedural clarity.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The patent replaces complex mechanical or chemical detection systems with a biological approach using antibodies. Instead of relying on physical or chemical properties of the modified base itself, the method uses the specific binding capability of antibodies to isolate and detect modified bases, simplifying the overall detection mechanism while improving reliability.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

3Productivity

If high-throughput sequencing is combined with affinity enrichment, then productivity and coverage of the transcriptome improve, but the cost and complexity of the protocol increase

Engineering Contradiction:

Improvethroughput of transcriptome characterizationVSAvoidcomplexity of sequencing protocol

Core Design Contradiction:

ProductivityVSDevice complexity

Solution Approach 1:

The patent merges two powerful technologies - affinity enrichment (immunoprecipitation) and high-throughput sequencing - into a unified workflow. This combination allows simultaneous achievement of high productivity and comprehensive transcriptome coverage. The merged protocol leverages the strengths of both methods: specific enrichment of modified base-containing transcripts and high-throughput identification of modification sites, while managing complexity through integrated protocol design.

Inventive Principle:

Principle #5Merging (Combining)

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 enables the identification of m6A sites across the transcriptome, revealing its prevalence, regulation, and functional roles, demonstrating that m6A is a widespread modification enriched near stop codons and in 3' UTRs, and provides insights into epigenetic regulation and potential associations with microRNA pathways.

Implementation Method 1

contacting a transcriptome comprising one or more modified bases with an antibody specific to the one or more modified bases under conditions effective to bind the antibody to the one or more modified bases

Methodology Applied

Scientific EffectAntibody binding:

Data Source

PatentUS11021703B2Methods and kit for characterizing the modified base status of a transcriptome

Publication Date: 2021.06.01 CORNELL UNIVERSITY

AI summary

This invention relates to a method of characterizing the modified base status of a transcriptome, which involves contacting a transcriptome comprising one or more modified bases with an antibody specific to the modified bases under conditions effective to bind the antibody to the modified bases; isolating, from the transcriptome, a pool of RNA transcripts to which the antibody binds; and identifying isolated RNA transcripts that are present in a higher abundance in the isolated pool relative to the transcriptome, where each of the isolated RNA transcripts that are present in a higher abundance in the isolated pool together characterize the modified base status of the transcriptome. Also disclosed are a method of diagnosis or prognosis of a disease, a method of determining the effect of a treatment on modified base levels in RNA, and a kit for characterizing the modified base status of a transcriptome.