smartSHAPE RNA Structure Detection with Minimal Input
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Solution Overview
Problem
Current RNA structure probing methods require large amounts of RNA, limiting their application to rare primary cells and tissue samples, and often fail to reflect the true functional states of cells due to deviations in cellular environments.
Innovation Solution
The development of smartSHAPE, a novel method that uses random RT and on-bead single-stranded DNA library construction to probe RNA structures with minimal RNA input, incorporating RNase I digestion and magnetic bead enrichment to remove background reverse transcription stop signals, allowing for accurate analysis of low-input RNA samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional RNA structure probing methods (icSHAPE, Structure-seq2) are used, then RNA structural information can be obtained, but a large amount of RNA input is required (approximately 10^7 cells)
Solution Approach 1:
The patent segments the reverse transcription process into multiple rounds with different primers (oligo-dT, random hexamers, and template-switching oligos). This segmentation allows progressive enrichment of modified RNA molecules while diluting background signals, enabling accurate structure probing with minimal RNA input (down to 10^4-10^5 cells) without requiring conventional large-scale RNA amounts
Solution Approach 2:
The patent performs preliminary chemical modification of RNA with SHAPE reagents before reverse transcription. This preliminary action creates detectable modifications at specific structural positions, allowing subsequent selective detection and enrichment of modified molecules during the multi-round RT process, thereby achieving high measurement precision with low RNA input
2Adaptability or versatility
If RNA structure probing is performed on rare primary cells and tissue samples, then biologically relevant RNA structures can be studied, but the limited RNA amount makes conventional methods inapplicable
Solution Approach 1:
The segmented multi-round reverse transcription approach with different primer sets allows the method to adapt to rare cell types by progressively enriching for modified RNA molecules. Each round selectively amplifies molecules with SHAPE modifications while controlling background, making the method versatile for studying rare primary cells and tissue samples that would provide insufficient RNA for conventional methods
Solution Approach 2:
The patent changes the reverse transcription parameters across multiple rounds (different primers, conditions, and cycling) to optimize enrichment of modified RNA from rare samples. By adjusting these parameters iteratively, the method achieves sufficient signal for analysis from minimal RNA inputs, enabling study of rare cell types that conventional methods cannot handle
3Ease of manufacture
If background reverse transcription stop signals are not removed, then the analysis process is simpler, but false positive signals from non-modification sites (e.g., endogenous modifications, G-quadruplexes) reduce accuracy
Solution Approach 1:
The patent extracts and removes background reverse transcription stop signals through a specific protocol step where unmodified RNA molecules are selectively removed or ignored. By taking out these background signals (caused by endogenous modifications like m1A, G-quadruplexes, or random RT shedding) through controlled RT conditions and bioinformatic filtering, the method maintains simplicity while significantly improving measurement precision
Solution Approach 2:
The patent converts the potentially harmful background RT stop signals into useful information by using them as negative controls. The multi-round RT process with different primers allows differentiation between true SHAPE modification signals and background signals. By analyzing the pattern of stops across rounds and comparing with control samples, the method transforms background noise into a means to identify and exclude false positives, thereby improving accuracy
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
smartSHAPE enables efficient and accurate analysis of whole transcriptome RNA secondary structures with as little as 1 ng of RNA, improving accuracy and reducing sample requirements, making it suitable for studying rare cells and tissues, including mammalian early embryos and patient biopsy samples.
Implementation Method 1
the background reverse transcription stop signals are removed by ribonuclease (RNase) digestion. More preferably, the background reverse transcription stop signals are removed by RNase I digestion.
Implementation Method 2
the cDNA enrichment is enrichment with magnetic beads; more preferably, the magnetic beads are streptavidin magnetic beads, such as MyOne C1 magnetic beads.
Implementation Method 3
RNA reverse transcription, removal of background reverse transcription stop signals caused by non-modification sites (premature RT stops)
Implementation Method 4
dimethyl sulfate (DMS) modifies single-stranded cytosines and adenines
Data Source
AI summary
Provided in the present invention are a method for detecting an RNA structure and the use thereof. According to the present invention, the step of removing the background of reverse transcription termination signals is included in the method for detecting an RNA structure, and false positive signals in a structural score calculation are reduced, and therefore the accuracy of the detection method is improved.