Small RNA Processing via Stem-Loop Primer and TSO

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Solution Overview

Problem

Current methods for processing RNA molecules are limited in their ability to efficiently convert RNA into complementary DNA (cDNA) while minimizing interference from cellular RNAs and maintaining sensitivity for detecting small RNA molecules.

Innovation Solution

A method involving a reaction mixture with a stem-loop primer and a template switch oligonucleotide (TSO) that provides an exogenous template for reverse transcription, allowing for the efficient generation of cDNA from RNA molecules, even in the presence of cellular RNAs.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If conventional reverse transcription methods are used, then RNA can be converted to cDNA, but cellular RNAs interfere with the detection sensitivity and specificity of small RNA molecules

Engineering Contradiction:

Improvedetection sensitivityVSAvoidinterference from cellular RNAs

Core Design Contradiction:

Measurement precisionVSObject-affected harmful factors

Solution Approach 1:

The patent employs a template switch oligonucleotide (TSO) as an intermediary element that facilitates selective reverse transcription of small RNAs. The TSO contains a template switching sequence that binds to the 3' end of small RNAs, enabling cDNA synthesis specific to small RNA molecules while excluding cellular RNAs from the reaction, thus resolving the interference issue without sacrificing detection sensitivity

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The stem-loop primer is designed with localized specificity through its hemiprobe segments that hybridize to specific end regions of small RNAs. This local quality approach ensures that the primer binds selectively to small RNA structures rather than cellular RNAs, achieving both high detection sensitivity and specificity by targeting the unique structural features of small RNAs

Inventive Principle:

Principle #3Local quality

2Productivity

If standard reverse transcription is used without template switching, then the process is simpler, but the conversion efficiency of small RNAs is insufficient

Engineering Contradiction:

Improveconversion efficiencyVSAvoidreaction mixture complexity

Core Design Contradiction:

ProductivityVSDevice complexity

Solution Approach 1:

The template switch oligonucleotide performs preliminary action by providing a pre-configured template structure that guides the reverse transcription process. The TSO's template switching sequence is pre-designed to bind to the 3' end of small RNAs, ensuring efficient cDNA synthesis from the outset. This preliminary preparation increases conversion efficiency without requiring complex multi-step procedures, as the TSO automatically directs the reverse transcription enzyme to the correct template

Inventive Principle:

Principle #10Preliminary action

3Reliability

If cellular RNAs are present in the reaction, then the reaction mixture is more complex, but the ability to detect small RNA molecules is compromised

Engineering Contradiction:

Improvespecificity of small RNA detectionVSAvoidpresence of cellular RNAs

Core Design Contradiction:

ReliabilityVSQuantity of substance

Solution Approach 1:

The patent effectively extracts or removes cellular RNAs from the detection process by using the template switch oligonucleotide to selectively target only small RNA molecules. The TSO's template switching mechanism ensures that reverse transcription occurs exclusively on small RNAs, thereby taking out cellular RNAs from the productive reaction pathway. This extraction approach maintains high reliability in small RNA detection while accommodating the presence of cellular RNAs in the reaction mixture

Inventive Principle:

Principle #2Taking out (Extraction)

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 method effectively converts RNA into cDNA, enabling sensitive detection of small RNA molecules while overcoming interference from cellular RNAs, thus improving the efficiency and specificity of RNA processing.

Implementation Method 1

a 5' hemiprobe segment configured to hybridize to a first end region of the RNA molecule; a stem-loop segment; and a 3' hemiprobe segment configured to hybridize to a second end region of the RNA molecule

Methodology Applied

Scientific EffectHybridization:

Implementation Method 2

providing a reaction mixture configured to produce a complementary deoxyribonucleic acid (cDNA) molecule from the RNA molecule

Methodology Applied

Scientific EffectReverse transcription:

Implementation Method 3

a template switch oligonucleotide (TSO) that provides an exogenous template, wherein the TSO is configured for elongation of the RNA molecule when the RNA molecule is reverse-transcribed

Methodology Applied

Scientific EffectTemplate switching:

Data Source

PatentUS20250163504A1Processing method for small rnas

Publication Date: 2025.05.22 TATAA BIOCENT

AI summary

Methods, compositions, and systems for processing small RNAs are described herein.