Oligonucleotide-Guided RNA Cleavage for mRNA End Analysis
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Solution Overview
Problem
Existing methods for analyzing the 5′ and 3′ terminal regions of mRNA molecules are inefficient, costly, and not applicable to a wide range of mRNA products with different sequences or structures, and lack the ability to simultaneously assess both regions for quality control in pharmaceutical RNA production.
Innovation Solution
A method involving conjugates with oligonucleotides and chemical moieties with RNA cleaving activity is used to sequence-specifically cleave mRNA molecules, allowing for the analysis of 5′ and 3′ fragments through hybridization, followed by separation and analysis using techniques like HPLC and mass spectrometry.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If conventional methods are used to analyze 5' and 3' terminal regions of mRNA, then analysis can be performed, but the methods are inefficient, costly, and not applicable to a wide range of mRNA products
Solution Approach 1:
The patent employs a universal conjugate design where a single chemical moiety with RNA cleaving activity can be directed to different target sequences through various oligonucleotide sequences. This allows the same core chemical structure to analyze diverse mRNA products including different cap structures (Cap0, Cap1, Cap2) and polyA tail configurations, eliminating the need for product-specific reagents while maintaining high analysis efficiency through standardized HPLC and mass spectrometry workflows
Solution Approach 2:
The patent segments the mRNA molecule into analyzable fragments by using conjugates that cleave at specific positions near the 5' or 3' ends. By designing oligonucleotides with complementary sequences to target regions, the conjugates generate discrete 5' fragments or 3' fragments that can be separately analyzed. This segmentation enables simultaneous assessment of both terminal regions through a single analytical workflow, improving both versatility and efficiency
2Measurement precision
If existing analysis methods are used, then some quality parameters can be assessed, but the methods lack the ability to simultaneously assess both 5' and 3' regions
Solution Approach 1:
The patent merges the analysis of 5' and 3' terminal regions into a single integrated workflow. By using conjugates that can be applied to the entire mRNA population simultaneously, both terminal regions are cleaved and generated fragments are analyzed together through HPLC and mass spectrometry. This combining approach enables comprehensive quality control assessing cap structures, polyA tail lengths, orientations, and methylation patterns in one unified process, improving measurement precision without proportionally increasing device complexity
Solution Approach 2:
The patent introduces conjugates as intermediary agents that mediate the cleavage process. These conjugates consist of a chemical moiety with RNA cleaving activity linked to an oligonucleotide sequence that directs the cleavage to specific locations. The conjugate acts as a mediator between the analytical system and the mRNA molecule, enabling precise and simultaneous assessment of both terminal regions while simplifying the overall analysis protocol through standardized reagent application
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 provides a precise, cost-effective, and efficient analysis of mRNA quality, capable of distinguishing between different cap structures and polyA tail lengths, orientations, and methylation patterns, suitable for a broad range of mRNA products.
Implementation Method 1
The sequence of said oligonucleotide is complementary to a target sequence of the RNA molecule to be analyzed
Implementation Method 2
chemical moiety with RNA cleaving activity to obtain RNA fragments
Data Source
AI summary
The present invention is concerned with methods for analyzing RNA molecules. The provided methods involve conjugates for RNA cleavage comprising a chemical moiety with RNA cleaving activity and an oligonucleotide. The oligonucleotide is designed based on a target sequence present in an RNA molecule, and the cleavage of the RNA molecule is inter alia carried out at conditions allowing the hybridization of the oligonucleotide to the target 5 sequence. Thereby, the method is easily applicable to RNA molecules of any sequence. The method further involves the analysis of the RNA fragments obtained after cleavage to obtain information on the physical properties of the RNA molecule.