Single-Molecule RNA Structure Profiling via smStructure-seq
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Solution Overview
Problem
Current methods for determining RNA structure in vivo face challenges such as distinguishing RNA isoform structure heterogeneity, achieving single-molecule resolution, and dissecting RNA structure conformation heterogeneity, with existing technologies like short read sequencing and nanopore-based methods falling short in accuracy and resolution.
Innovation Solution
The development of a novel single-molecule structure sequencing method (smStructure-seq) combined with a new analysis pipeline called DaVinci, which uses high-accuracy single-molecule sequencing and stochastic context-free grammar to directly cluster structural information from mutation profiles, enabling accurate estimation of RNA structure conformations at the single-molecule level.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If short read sequencing platforms are used for RNA structure analysis, then high throughput genome-wide RNA structure analyses can be enabled, but RNA structure information within shared regions between isoforms cannot be distinguished
Solution Approach 1:
The patent segments the RNA structure analysis problem by developing isoform-specific chemical probing methods that can distinguish structure information from different isoforms in shared regions. This is achieved through techniques such as isoform-specific mutational profiling and long-read sequencing that resolve individual isoform structures rather than providing averaged data across all isoforms.
Solution Approach 2:
The patent transitions from short-read sequencing (1D linear fragments) to long-read sequencing (1D full-length transcripts) and subsequently to single-molecule resolution (adding the dimension of individual molecule analysis). This dimensional change enables the distinction of RNA structure information at the level of individual isoforms and even individual molecules, resolving the limitation of shared region analysis.
2Quantity of substance
If nanopore-based methods are used for single-molecule sequencing, then single molecule reads can be obtained, but the accuracy is only 86% and true single-molecule resolution of RNA structure information cannot be achieved
Solution Approach 1:
The patent merges multiple sequencing technologies and chemical probing methods to achieve both single-molecule resolution and high accuracy. By combining long-read sequencing with chemical modification data (such as DMS, SHAPE, or CMCT probing) and mutational profiling, the method achieves accurate RNA structure determination at the single-molecule level, overcoming the limitations of using any single method alone.
Solution Approach 2:
The patent changes the accuracy parameter by using high-fidelity polymerases and multiple passes of sequencing (circular consensus sequencing) to achieve >99% accuracy. It also changes the resolution parameter by using long-read sequencing technologies that can read full-length transcripts, thereby resolving individual isoform structures with high accuracy at the single-molecule level.
3Ease of operation
If chemical reactivity-based clustering methods are used for RNA structure analysis, then computational analysis can be performed, but the methods generate clusters with extremely high or low chemical modifications that do not directly represent RNA structure conformations
Solution Approach 1:
The patent introduces chemical modification data (such as DMS, SHAPE, or CMCT probing results) as an intermediary that provides direct experimental evidence of RNA structure. These chemical probes selectively modify accessible nucleotides based on their structural context (single-stranded vs. double-stranded), creating a molecular-level map of RNA structure that serves as a mediator between the RNA molecule and computational analysis, thereby accurately representing true structural conformations.
Solution Approach 2:
The patent replaces traditional mechanical clustering approaches (which group data based on overall similarity) with a chemistry-based system where chemical probes directly interact with RNA structures. This substitution allows the system to detect and represent RNA conformations through chemical reactivity patterns rather than computational clustering, providing more accurate and direct structural 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 approach allows for the simultaneous addressing of heterogeneities in RNA isoforms and structural conformations, achieving true single-molecule resolution and providing accurate RNA structure information that was previously unattainable.
Implementation Method 1
subjecting a population of RNA molecules to structure-specific chemical treatments where individual RNA molecules will be modified
Implementation Method 2
reverse-transcribing the modified RNA to provide the corresponding complementary DNA (cDNA) molecule
Data Source
AI summary
Described is a method for determining structure of an RNA molecule, the method comprising: a) subjecting a population of RNA molecules to structure-specific chemical modifications such that individual RNA molecules are modified; b) reverse-transcribing the modified RNA to provide a complementary DNA (cDNA) molecule, and generating double stranded DNA from said cDNA molecule; c) performing single-molecule sequencing of the double stranded cDNA using a sequencing format which provides multiple reads of each molecule to arrive at a consensus sequence representing a chemical mutation profile for an individual DNA; d) using said chemical mutation profile to determine likelihood of an RNA molecule being single stranded or double stranded at each individual base, to thereby determine the structure of the RNA molecule.