Rolling Circle Reverse Transcription for Low-Abundance circRNA Sequencing

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Solution Overview

Problem

Existing methods for detecting and sequencing circular RNAs (circRNAs) are inefficient, require purification steps that diminish low concentrations, and suffer from sequencing bias, making it difficult to analyze small or large circRNAs in complex biological samples.

Innovation Solution

Utilizing Group II Intron reverse transcriptase (Intron-RT) for rolling circle reverse transcription to generate concatemeric cDNA from circRNAs, which can be sequenced accurately without prior purification, enhancing sensitivity and reducing bias.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If existing methods for detecting and sequencing circRNAs are used, then circRNA detection can be performed, but the methods are inefficient and require purification steps that diminish low concentrations of circRNAs

Engineering Contradiction:

Improvedetection efficiencyVSAvoidcircRNA concentration

Core Design Contradiction:

ProductivityVSQuantity of substance

Solution Approach 1:

The patent applies preliminary action by performing rolling circle reverse transcription directly on circRNAs in complex biological samples without prior purification. The method prepares the circRNAs for sequencing by generating concatemeric cDNA in situ, eliminating the need for separate purification steps that would otherwise diminish the already low concentrations of circRNAs.

Inventive Principle:

Principle #10Preliminary action

2Measurement precision

If existing sequencing methods are used, then circRNA sequencing can be performed, but the methods suffer from sequencing bias

Engineering Contradiction:

Improvesequencing accuracyVSAvoidsequencing reliability

Core Design Contradiction:

Measurement precisionVSReliability

Solution Approach 1:

The patent replaces conventional PCR-based amplification methods with rolling circle reverse transcription. This substitution eliminates the amplification bias inherent in traditional methods by using a template-directed synthesis mechanism that faithfully replicates the circRNA sequence without preferential amplification of certain regions, thereby improving both accuracy and reliability.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

3Measurement precision

If purification steps are performed to concentrate circRNAs, then detection sensitivity may improve, but the process diminishes low concentrations of circRNAs

Engineering Contradiction:

Improvedetection sensitivityVSAvoidcircRNA loss

Core Design Contradiction:

Measurement precisionVSLoss of substance

Solution Approach 1:

The method applies self-service by enabling the circRNAs to serve their own amplification and concentration needs through rolling circle reverse transcription. The circRNAs act as templates for generating multiple copies of cDNA in a self-amplifying process, eliminating the need for external purification and concentration steps that would cause substance loss.

Inventive Principle:

Principle #25Self-service

4Productivity

If conventional reverse transcription methods are used, then cDNA can be synthesized, but the methods are inefficient for circRNA detection

Engineering Contradiction:

ImprovecDNA synthesis efficiencyVSAvoiddetection accuracy

Core Design Contradiction:

ProductivityVSMeasurement precision

Solution Approach 1:

The patent implements continuity of useful action through rolling circle reverse transcription, where the reverse transcriptase continuously synthesizes cDNA around the circular circRNA template without dissociation. This continuous synthesis generates long concatemeric cDNA molecules efficiently, improving both the productivity of cDNA synthesis and the accuracy of circRNA detection compared to conventional discontinuous methods.

Inventive Principle:

Principle #20Continuity of useful action

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 method provides a sensitive and efficient means to detect and sequence circRNAs, achieving higher yields and accuracy, especially in complex biological samples, by forming concatemeric cDNA that can be sequenced directly or after amplification, thereby overcoming the limitations of existing techniques.

Implementation Method 1

Group II Intron reverse transcriptase (Intron-RT) for rolling circle reverse transcription to generate concatemeric cDNA from circRNAs

Methodology Applied

Scientific EffectReverse transcription:

Implementation Method 2

rolling circle reverse transcription of circRNA to form cDNA containing concatemers of sequence units that are complementary to the circRNA

Methodology Applied

Scientific EffectRolling circle amplification:

Data Source

PatentEP4137582B1Rolling circle reverse transcription of circular RNA

Publication Date: 2026.01.28 NEW ENGLAND BIOLABS INC

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Figure 2A~2B

AI summary

Compositions, methods and kits are provided that enable the detection, analysis and/or sequencing of small or large target RNA molecules whether synthetic, purified or within a biological fluid, or in cell lysate that may contain non-target RNA and other contaminating molecules without the need for depletion or purification steps that diminish what might already be low concentrations of the target molecule. The methods, compositions and kits rely on the use of a Group II Intron reverse transcriptase (Intron-RT) that have strand displacing properties and can generate concatemers in cDNA by rolling circle transcription of circRNAs that may be naturally circular or circularized in vitro from linear RNA.