RNA Enrichment via 5' End Capping and Affinity Tagging
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Solution Overview
Problem
Current methods for rRNA depletion in RNA analysis are inefficient and require prior knowledge of ribosomal sequences, often leaving substantial rRNA and degraded RNA in the mixture.
Innovation Solution
A method involving the addition of a modified GMP to the 5′ end of 5′-diphosphorylated or 5′-triphosphorylated RNA molecules using a capping enzyme and an affinity tag-labeled GTP, followed by enrichment using an affinity matrix, to selectively isolate and sequence eukaryotic mRNA from a mixture of RNAs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If DNA oligonucleotides complementary to rRNA are hybridized and removed, then rRNA depletion is achieved, but customizing DNA sequence and requiring a priori knowledge of ribosomal sequence is needed
Solution Approach 1:
The invention changes the chemical parameter of the 5' end of RNA molecules by using enzymatic decapping to remove the 5' cap structure from eukaryotic mRNA, converting it to a 5' monophosphate form. This parameter change enables selective recognition and depletion of eukaryotic mRNA without requiring sequence-specific DNA probes, thereby resolving the contradiction between depletion efficiency and method complexity
Solution Approach 2:
The invention replaces the mechanical/chemical system of hybridization-based depletion with an enzymatic system. Instead of using DNA oligonucleotides that must hybridize to target sequences, the method uses enzymes (decapping enzyme and phosphorylase) that recognize and modify specific chemical structures (5' cap and phosphate groups), eliminating the need for custom DNA sequence design while maintaining high specificity
2Reliability
If enzymatic degradation of 5′ monophosphate RNA is used, then rRNA removal is attempted, but the reaction is inefficient and substantial rRNA and degraded RNA remain
Solution Approach 1:
The invention applies preliminary action by first converting all target RNA molecules to a uniform intermediate state (5' monophosphate) through decapping and phosphorylation, then applying the degradation enzyme. This preliminary preparation ensures that the degradation reaction acts on all target molecules efficiently, preventing the incomplete degradation observed when enzymes are applied directly to mixed RNA populations
Solution Approach 2:
The invention applies local quality by creating a specific chemical signature (5' monophosphate group) at the 5' end of target RNA molecules through enzymatic modification. This localized chemical modification serves as a recognition site for the degradation enzyme, ensuring selective and efficient degradation of only the intended targets while leaving other RNA molecules intact
3Reliability
If 5′-m7Gppp capped RNA is not decapped before analysis, then eukaryotic mRNA cannot be selectively enriched, but decapping requires specific enzymatic treatment
Solution Approach 1:
The invention introduces an intermediary chemical form (5' monophosphate) that serves as a universal recognition signal for downstream processing enzymes. By converting the diverse 5' end structures of RNA molecules to this intermediate form, the method creates a common platform that enables selective enrichment and analysis without requiring multiple different enzymatic treatments for different RNA types
Solution Approach 2:
The invention segments the RNA analysis process into distinct modular steps: decapping, phosphorylation, and degradation/enrichment. Each step uses a specific enzyme that acts on a particular chemical feature, allowing the complex task of eukaryotic mRNA enrichment to be broken down into manageable, highly specific operations that can be performed in sequence
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 enriches for eukaryotic mRNA by converting 5′-diphosphorylated or 5′-triphosphorylated RNA molecules into 5′-capped labeled RNA, allowing for efficient removal of rRNA and degraded RNA, thereby improving the accuracy of RNA sequencing and analysis.
Implementation Method 1
adding a modified GMP to the 5′ end of 5′-diphosphorylated or 5′-triphosphorylated RNA molecules in a sample by incubating the sample with a modified GTP and a capping enzyme
Implementation Method 2
enriching for RNA comprising the affinity tag-labeled GMP using an affinity matrix that binds to the affinity tag
Data Source
AI summary
A method of enriching for a population of RNA molecules in a mixture of RNAs is provided. In some embodiments, the method may comprise (a) adding an affinity tag to the 5′ end of 5′-diphosphorylated or 5′-triphosphorylated RNA molecules in a sample by incubating the sample with an affinity tag-labeled GTP and a capping enzyme; and (b) enriching for RNA comprising the affinity tag-labeled GMP using an affinity matrix that binds to the affinity tag.