5' Protection Dependent Amplification for RNA Integrity
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Solution Overview
Problem
Current methods for analyzing and selecting nucleic acids based on their 5' ends, such as mRNA, are either not very selective or involve multistep protocols that increase the risk of RNA degradation, leading to biases towards shorter RNA molecules and loss of full-length sequence information.
Innovation Solution
A method that involves reverse transcribing RNA into cDNA before performing potentially harmful steps, using 5' protecting groups like caps or triphosphates to preserve full-length information, and employing double strand-specific ligation to label cDNAs only when a 5' protecting group is present, thereby avoiding unnecessary enzymatic reactions that could degrade RNA.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If multistep protocols are used to select for mRNA based on 5' ends, then selectivity is improved, but RNA degradation increases and full-length sequence information is lost
Solution Approach 1:
The patent performs reverse transcription of RNA to cDNA before performing potentially harmful steps such as enzymatic reactions. By converting RNA to cDNA first, the original RNA is protected from degradation while still allowing selective processing of full-length molecules based on their 5' protecting groups.
Solution Approach 2:
The patent uses the cDNA molecule as an intermediary carrier that preserves the full-length sequence information of the original RNA. The cDNA serves as a stable intermediate that can undergo selective processing without degrading the original RNA template, thus maintaining both selectivity and RNA integrity.
2Measurement precision
If multiple enzymatic steps are performed to enrich for full-length RNA, then selectivity for full-length molecules is improved, but bias towards shorter RNA molecules is introduced
Solution Approach 1:
The patent performs reverse transcription to create cDNA copies before performing selective enrichment steps. This preliminary conversion allows subsequent enzymatic reactions to act on the stable cDNA rather than the original RNA, preventing degradation bias while maintaining full-length selection accuracy through cap-dependent processes.
Solution Approach 2:
The patent creates cDNA copies of the original RNA molecules as faithful replicas that preserve full-length sequence information. These copies can then be selectively processed and amplified without degrading the original RNA population, eliminating bias towards shorter molecules while maintaining accurate representation of full-length transcripts.
3Device complexity
If RNA is processed directly without reverse transcription, then the process is simpler, but the risk of RNA degradation increases
Solution Approach 1:
The patent performs reverse transcription as a preliminary step that converts labile RNA into stable cDNA before subsequent processing steps. Although this adds a step to the protocol, it dramatically reduces RNA degradation risk by eliminating the need to handle and process the original RNA through potentially harmful enzymatic reactions.
Solution Approach 2:
The cDNA serves as an intermediary that shields the original RNA from degradation. By performing all subsequent selective enrichment and processing steps on the cDNA copy rather than the original RNA, the protocol minimizes exposure of the RNA to harmful factors while preserving full-length sequence information.
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 preserves the full-length sequence information of RNA during analysis, reducing the risk of RNA degradation and enhancing selectivity in tagging specific 5' ends, while minimizing the introduction of biases towards shorter RNA molecules.
Implementation Method 1
b1) annealing at least one oligonucleotide primer to the template strand of said RNA and potentially other nucleic acids, and template sequence dependent extending said primer, thereby obtaining a complementary nucleic acid strand annealed to its template strand
Implementation Method 2
5' protection dependent amplification... RNA comprising a 5' protecting group, such as a 5' cap and/or 5' polyphosphate structure
Implementation Method 3
labelling a complementary nucleic acid of the RNA, thereby specifically generating a labelled nucleic acid complementary to an RNA at least originally comprising a 5' protecting group
Data Source
AI summary
The present invention relates to methods for generating a labelled nucleic acid from an RNA comprising a 5′ protecting group, said method comprises the steps of obtaining a mixture of template strands of nucleic acids, said mixture comprising said RNA and further potentially other nucleic acids without a 5′ protecting group, annealing at least one oligonucleotide primer to the template strand of said RNA and potentially other nucleic acids, and template sequence dependent extending said primer, thereby obtaining a complementary nucleic acid strand annealed to its template strand, or providing the RNA in duplex with a complementary nucleic acid strand annealed to its template strand, andoptionally modifying the extension product of said nucleic acids without 5′ protecting group either on the 5′ end of the template strand or on the 3′ end of the complementary strand, or both, and labelling a complementary nucleic acid of a double stranded nucleic acid not modified, wherein therefore the labelled nucleic acid did have a 5′ protecting group on the RNA template strand, said 5′ protecting group being optionally removed after modification, and/or labelling a complementary nucleic acid of a double strand dependent on the presence of the 5′ protecting group on the complementary nucleic acids template RNA strand by double strand dependent ligation, thereby specifically generating a labelled nucleic acid complementary to an RNA at least originally comprising a 5′ protecting group.