Primer-Protected RNA Mapping for Complete Long-Sequence Coverage
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Solution Overview
Problem
Current RNA mapping processes are inadequate for accurately sequencing long RNA molecules, such as mRNA, due to their length and complexity, leading to incomplete mappings and challenges in distinguishing modified nucleotides.
Innovation Solution
A method involving hybridization of a primer with an RNA molecule, followed by enzymatic digestion to form fragments, and analysis using liquid chromatography-mass spectrometry, with optimized conditions and enzyme selection to ensure complete sequence mapping.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If current RNA mapping processes are used on long RNA molecules, then the process is simple, but the sequence coverage is incomplete and mapping accuracy is poor
Solution Approach 1:
The long RNA molecule is divided into multiple shorter fragments through controlled enzymatic digestion. Primers are designed to bind to specific regions and protect them from digestion, creating a segmentation strategy that enables complete sequence coverage. The RNA is digested into fragments of manageable size (e.g., 6-20 nucleotides) that can be accurately analyzed by mass spectrometry, while primers protect specific regions to ensure all segments are mapped.
Solution Approach 2:
Primers are hybridized to the RNA molecule before digestion to pre-establish protected regions. This preliminary action ensures that when enzymatic digestion occurs, specific segments are protected from cleavage, allowing systematic coverage of the entire RNA sequence. The primers are strategically designed and bound in advance to guide the digestion process toward complete sequence mapping.
2Measurement precision
If enzymatic digestion is performed without primers, then the process is simple, but the RNA molecule cannot be adequately protected and sequence mapping is incomplete
Solution Approach 1:
Primers serve as intermediary molecules that mediate between the RNA target and the enzymatic digestion process. These short oligonucleotide sequences bind to complementary regions on the RNA molecule, acting as protective intermediaries that prevent enzyme access to specific sites. The primers translate the complex task of protecting entire long RNA molecules into manageable protected segments, enabling complete sequence coverage through systematic digestion of unprotected regions.
3Measurement precision
If multiple primers are used to increase coverage, then sequence mapping improves, but the hybridization process becomes more complex
Solution Approach 1:
A set of primers is designed with universal characteristics that enable them to work together in a single hybridization mixture. Each primer is optimized to bind to specific regions while maintaining consistent structural and thermal properties. This universality allows multiple primers to be combined in equimolar ratios, simplifying the hybridization operation while achieving comprehensive sequence coverage through the collective action of all primers in the set.
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 accurate sequencing of long RNA molecules by forming stable RNA/primer hybrids, allowing for complete digestion and analysis of RNA fragments, thereby confirming the desired therapeutic RNA sequence.
Implementation Method 1
hybridizing a primer with a portion of the RNA molecule to form an RNA/primer hybrid
Implementation Method 2
an enzyme configured to cleave a motif within the unprotected RNA region of the RNA/primer hybrid thereby forming two or more RNA fragments
Implementation Method 3
analyzing the two or more RNA fragments using liquid chromatography-mass spectrometry
Implementation Method 4
analyzing the two or more RNA fragments using liquid chromatography-mass spectrometry to determine a sequence of nucleotides in the RNA molecule
Data Source
AI summary
Disclosed herein are methods of mapping a sequence of an RNA molecule comprising the steps of hybridizing a protecting primer with a portion the RNA molecule to form RNA/primer hybrid, combining the RNA molecule, the primer, and a digestion assay, digesting the RNA molecule hybridized with the primer in the digestion assay, and analyzing the two or more RNA fragments using liquid chromatography-mass spectrometry to determine the sequence of nucleotides in the RNA molecule. Also disclosed herein are digestion assays for selectively cleaving an RNA molecule into two or more RNA fragments.