Ribosome Splitting for Non-ribosomal RNA Sample Production

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Solution Overview

Problem

Conventional ribosome profiling and RNA-seq methods are hindered by excessive ribosomal RNA (rRNA) contamination, which reduces the percentage of sequence reads available for analysis, particularly in protein coding regions, and existing rRNA-depletion methods are insufficient in reducing rRNA sequence reads.

Innovation Solution

A method involving the splitting of ribosomal subunits and mRNAs using a chelating agent, followed by ultrafiltration to remove the ribosomal subunits, thereby producing a non-ribosomal RNA-containing sample that reduces rRNA content, allowing for more efficient analysis of mRNA and non-coding RNAs.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Quantity of substance

If conventional ribosome profiling or RNA-seq methods are used, then sequencing can be performed, but rRNA excessively occupies the sequencing library resulting in low percentage of sequence reads available for analysis

Engineering Contradiction:

Improvepercentage of sequence reads available for analysisVSAvoidrRNA contamination

Core Design Contradiction:

Quantity of substanceVSObject-generated harmful factors

Solution Approach 1:

The ribosome is segmented into large and small subunits by adding a chelating agent that removes magnesium ions, causing the ribosome to dissociate. This segmentation allows the mRNA to be separated from the ribosomal components, enabling subsequent removal of the subunits and enrichment of non-ribosomal RNA for sequencing analysis.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The harmful rRNA component is extracted from the sequencing library by removing ribosomal subunits through ultrafiltration or other separation methods after ribosome dissociation. This extraction eliminates the source of rRNA contamination, increasing the proportion of useful sequence reads for mRNA and non-coding RNA analysis.

Inventive Principle:

Principle #2Taking out (Extraction)

2Quantity of substance

If rRNA-subtraction oligonucleotides are used to reduce rRNA sequence reads, then some rRNA depletion is achieved, but the method is insufficient in reducing rRNA sequence reads to acceptable levels

Engineering Contradiction:

ImproverRNA sequence readsVSAvoideffectiveness of rRNA depletion

Core Design Contradiction:

Quantity of substanceVSReliability

Solution Approach 1:

The ribosome is dissociated into subunits before sequencing library preparation, preventing rRNA from being incorporated into the library in the first place. This preliminary action is more effective than post-library rRNA subtraction methods, as it addresses the root cause of rRNA presence in sequencing data.

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The magnesium ion concentration is changed by adding a chelating agent, which alters the stability of ribosome-mRNA complexes. This parameter change causes ribosome dissociation and releases mRNA, enabling effective separation and depletion of rRNA before sequencing.

Inventive Principle:

Principle #35Parameter changes

3Productivity

If ribosomes are not removed from the sample, then the sample can be prepared quickly, but rRNA contamination significantly reduces the efficiency of transcriptome analysis

Engineering Contradiction:

Improveefficiency of transcriptome analysisVSAvoidcomplexity of sample preparation method

Core Design Contradiction:

ProductivityVSDevice complexity

Solution Approach 1:

The ribosome is segmented into large and small subunits through chelating agent treatment, which simplifies the subsequent separation process. The segmented subunits can be easily removed by ultrafiltration or other size-based separation methods, improving transcriptome analysis efficiency without excessive complexity.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

A chelating agent is introduced as an intermediary substance to mediate the dissociation of ribosomes. This intermediary facilitates the separation of mRNA from ribosomal components by removing magnesium ions, enabling effective rRNA depletion while maintaining a relatively simple procedural framework.

Inventive Principle:

Principle #24Intermediary (Mediator)

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 significantly increases the percentage of mRNA and non-coding RNA reads, improving the efficiency of transcriptome analysis by reducing rRNA contamination to 23% or less, and can be combined with rRNA-subtraction oligonucleotides to further reduce rRNA reads to 10% or less.

Implementation Method 1

splitting of ribosomal subunits and mRNAs using a chelating agent

Methodology Applied

Scientific EffectChelation:

Implementation Method 2

followed by ultrafiltration to remove the ribosomal subunits

Methodology Applied

Scientific EffectUltrafiltration: Semipermeable Membrane

Data Source

PatentUS20230002755A1Method for producing non-ribosomal RNA-containing sample

Publication Date: 2023.01.05 RIKEN CO LTD

AI summary

An object of the present invention is to provide a method for producing a non-ribosomal RNA-containing sample, which comprises a novel step for removing ribosomes. According to the present invention, there is provided a method for producing a non-ribosomal RNA-containing sample, which comprises the step (a) of splitting subunits of ribosomes and mRNAs in a sample containing mRNAs and ribosomes, and the step (b) of removing the subunits of ribosomes split in the step (a).