Circular RNA Amplification via cDNA Circularization
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Solution Overview
Problem
Current methods fail to effectively enrich and amplify circular RNA molecules due to their low abundance and similar sequence homology with linear RNAs, leading to incomplete cDNA copies that lose the circular structure, making it difficult to identify original circular RNA sequences.
Innovation Solution
The method involves priming circular RNA templates with DNA primers and extending them with reverse transcriptase to generate multiple cDNA copies, followed by ligating linear cDNA fragments to form covalently closed circular cDNA molecules, using enzymes like T4 DNA ligase and RNase H to maintain the circular structure and enhance amplification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If ribosomal RNA depletion or exoribonuclease digestion is used to enrich circular RNA, then the ratio of circular RNA to other species increases, but large amounts of RNA material (20 to 60 μg) are required making the technique impractical
Solution Approach 1:
The patent performs preliminary circularization of linear cDNA before amplification. By converting linear cDNA to circular cDNA early in the process, the subsequent rolling circle amplification can proceed efficiently with minimal input material, resolving the contradiction between enrichment effectiveness and material requirement
Solution Approach 2:
The patent uses rolling circle amplification to generate multiple copies of circular cDNA from a single circular template. This copying mechanism allows amplification of circular RNA sequences without requiring large amounts of starting material, as each circular template can produce numerous identical copies
2Productivity
If standard cDNA synthesis and amplification methods are used, then linear RNA is amplified, but the circular structure is lost making it difficult to identify original circular RNA sequences
Solution Approach 1:
The patent performs preliminary circularization of cDNA before amplification by ligating the 5' and 3' ends of linear cDNA. This preserves the circular structure information throughout the amplification process, allowing identification of original circular RNA sequences while maintaining high amplification efficiency through rolling circle replication
Solution Approach 2:
The patent uses circular cDNA as an intermediary between circular RNA and amplified products. The circular cDNA serves as a stable template that maintains structural information while enabling high-yield amplification, thus preventing loss of circular structure information during the amplification process
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 allows for the preferential amplification of circular RNA over linear RNA, enabling more sensitive detection and cost-effective next-generation sequencing by generating accurate, multiple copies of circular RNA sequences while retaining the original structure.
Implementation Method 1
extending them with reverse transcriptase to generate multiple cDNA copies
Implementation Method 2
ligating linear cDNA fragments to form covalently closed circular cDNA molecules, using enzymes like T4 DNA ligase
Implementation Method 3
using enzymes like T4 DNA ligase and RNase H to maintain the circular structure
Data Source
AI summary
Provided herein are methods of amplifying nucleic acids. In particular, methods are provided for amplifying circular RNA molecules. In certain embodiments, circular DNA molecules for amplification are generated from circular RNA molecules.