RNA Sequencing via LC-MS and End Labeling
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Solution Overview
Problem
Current methods for nucleic acid sequencing, particularly RNA sequencing, face challenges such as impurities and non-specific fragmentation, which obscure true mass data and hinder efficient sequencing of RNA molecules, especially in biological samples, and lack effective tools for identifying and quantifying RNA modifications associated with diseases like breast cancer, type-2 diabetes, and obesity.
Innovation Solution
A direct liquid chromatography-mass spectrometry (LC-MS) based technique that sequences RNA without prior cDNA synthesis, allowing single nucleotide resolution and detection of RNA modifications by labeling the 5′ and/or 3′ ends of RNA, followed by random degradation and physical separation of RNA fragments for MS analysis, using affinity interactions and hydrophobic tags to enhance data analysis and distinguish between 5′ and 3′ ladder fragments.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of information
If RNA is subjected to degradation to generate mass ladders for sequencing, then sequence information can be obtained, but impurities and non-specific fragments are generated that obscure true mass data
Solution Approach 1:
The patent extracts and removes impurities and non-specific fragments from the degradation mixture through purification steps, isolating only the desired mass ladder fragments for sequencing analysis, thereby resolving the contradiction between obtaining sequence information and maintaining mass data accuracy
Solution Approach 2:
The patent introduces purification intermediaries (such as size exclusion chromatography media or other purification agents) that selectively bind or separate the desired RNA mass ladders from impurities and non-specific fragments, enabling accurate mass measurement while preserving sequence information
2Quantity of substance
If multiple cuts occur on each RNA strand during degradation, then more fragments are generated for analysis, but data complexity increases and analysis becomes difficult
Solution Approach 1:
The patent applies controlled degradation conditions that create a non-uniform distribution of cuts, with a higher frequency of single cuts near the ends of RNA strands and fewer internal cuts, generating sufficient fragments for sequencing while minimizing the complexity of internal fragment analysis
Solution Approach 2:
The patent performs preliminary purification and selection steps before analysis to enrich for ladder fragments with single cuts and remove or separate internal fragments with multiple cuts, simplifying downstream data analysis while maintaining sufficient fragment quantity for complete sequencing
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
Enables efficient sequencing of purified and mixed RNA samples, including those from biological samples, with improved accuracy in identifying and quantifying RNA modifications, facilitating the correlation of RNA functions with modifications and quality control of RNA-based therapeutics.
Implementation Method 1
liquid chromatography-mass spectrometry (LC-MS) based technique
Implementation Method 2
liquid chromatography-mass spectrometry (LC-MS) based technique
Implementation Method 3
using affinity interactions and hydrophobic tags to enhance data analysis and distinguish between 5′ and 3′ ladder fragments
Implementation Method 4
using affinity interactions and hydrophobic tags to enhance data analysis and distinguish between 5′ and 3′ ladder fragments
Data Source
AI summary
The present disclosure relates generally to novel methods for nucleic acid sequencing. Specifically, the invention relates to a liquid chromatography-mass-spectrometry (LC-MS) based technique for direct sequencing of RNA without cDNA. The technique allows one to simultaneously read an RNA sequence with single nucleotide resolution while determining the presence, type and location of a wide spectrum of RNA modifications.